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Adverse Effects of Immunosuppressant Drugs upon Airway Epithelial Cell and

Mucociliary Clearance: Implications for Lung Transplant Recipients

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Drugs (2013) 73:1157–1169
DOI 10.1007/s40265-013-0089-0
REVIEW ARTICLE
Adverse Effects of Immunosuppressant Drugs upon Airway
Epithelial Cell and Mucociliary Clearance: Implications for Lung
Transplant Recipients
Rogerio Pazetti • Paulo Manuel Pêgo-Fernandes •
Fabio Biscegli Jatene
Published online: 11 July 2013
Ó Springer International Publishing Switzerland 2013
Abstract Optimal post-transplantation immunosuppres-
sion is critical to the survival of the graft and the patient
after lung transplantation. Immunosuppressant agents tar-
get various aspects of the immune system to maximize
graft tolerance while minimizing medication toxicities and
side effects. The vast majority of patients receive mainte-
nance immunosuppressive therapy consisting of a triple-
drug regimen including a calcineurin inhibitor, a cell cycle
inhibitor and a corticosteroid. Although these immuno-
suppressant drugs are frequently used after transplantation
and to control inflammatory processes, limited data are
available with regard to their effects on cells other than
those from the immunological system. Notably, the airway
epithelial cell is of interest because it may contribute to
development of bronchiolitis obliterans through production
of pro-inflammatory cytokines. This review focuses the
current armamentarium of immunosuppressant drugs used
after lung transplantation and their main side effects upon
airway epithelial cells and mucociliary clearance.
1 Introduction
Lung transplantation is a lifesaving intervention for
patients with end-stage diseases of the lungs and the pul-
monary circulation. Several factors have been improved
since the first transplantation in 1963, such as optimal
patient selection, donor care, histocompatibility testing,
R. Pazetti (&)
P. M. Pêgo-Fernandes
F. B. Jatene
Laboratory of Thoracic Surgery Research-LIM61, Department of
Cardiopneumology, Heart Institute (InCor), Hospital das
Clı́nicas da Faculdade de Medicina da Universidade de São
Paulo, Avenida Doutor Arnaldo, 455, 1o. Andar, Sala 1220,
Pacaembu, São Paulo, SP 01246-000, Brazil
e-mail: rogeriopazetti@yahoo.com.br
graft preservation, surgical techniques, postoperative
management, and immunosuppressive therapy. All of these
factors have contributed significantly to the large increases
in the numbers of lung transplantation procedures being
performed in adults each year—from 5 in 1985 to 3,519 in
2010 [1].
The vast majority of patients receive maintenance
immunosuppressive therapy consisting of a triple-drug
regimen. Between 2002 and 2010, the maintenance regi-
men of approximately 95 % of lung transplant recipients at
both 1 and 5 years after transplantation consisted of a
calcineurin inhibitor plus a cell cycle inhibitor. More pre-
cisely, the combination of tacrolimus and mycophenolate
mofetil has now become the most widely used combination
internationally (Table 1) [1].
Optimal post-transplantation immunosuppression is
critical to the survival of the graft and the patient. How-
ever, these medications also contribute to significant mor-
bidity and mortality in the lung transplant population.
Serious complications that occur as a result of chronic
immunosuppression include malignancies, neuro- and
hepatotoxicity, lymphoproliferative disorders, renal failure,
thrombocytopenia, hypertension and opportunistic infec-
tions (Table 2) [2].
Infectious complications are one of the most common
causes of morbidity and mortality at all timepoints after
lung transplantation (Table 3) [1], and at least 65 % of the
infections involve the respiratory tract [3]. Possible reasons
for this very high incidence of infectious complications
include an allograft continuously exposed to the environ-
ment, bronchial anastomotic problems, and impaired mu-
cociliary clearance [4].
Mucociliary clearance is an essential innate immune
protective mechanism in the airways. A coordinated system
of epithelial water and ion transport, mucin secretion, cilia
1158 R. Pazetti et al.

Table 1 Maintenance immunosuppressive agents commonly used and damage. This disruption may be due to diseases like
after lung transplantation [1] primary ciliary dyskinesia, cystic fibrosis and asthma, or
Drug class Agent Percentage of may be secondary to factors such as pollutant exposure and
patients drug toxicity effects [5].
At At Although these immunosuppressant drugs are frequently
1 year 5 years used after transplantation and to control inflammatory
processes, limited data are available with regard to their
Calcineurin Cyclosporine 16 19
inhibitor
effects on cells other than those from the immunological
Tacrolimus 83 77
system. Airway epithelial cells are of special interest
Cell cycle Azathioprine 30 31
because they may contribute to development of bronchi-
inhibitor Mycophenolate mofetil 60 53 olitis obliterans syndrome (BOS) through production of
Combination Cyclosporine ? azathioprine 5 6 pro-inflammatory cytokines [6]. BOS is the major clinical
Cyclosporine ? mycophenolate 7 8 manifestation of chronic lung allograft rejection [7] and the
mofetil
main cause of mortality (causing about 27 % of deaths)
Tacrolimus ? azathioprine 22 20
between 1 and 10 years after lung transplantation [1].
Tacrolimus ? mycophenolate 46 36
mofetil
Acute pulmonary allograft rejection is the principal risk
factor for development of BOS, but infection and fibro-
proliferation are also considered important risk factors for
Table 2 Main side effects of maintenance immunosuppressive this process [7]. BOS is characterized by an intense
agents inflammatory process with fibrosis and extracellular matrix
Agent Characteristic side effects deposition in the subepithelium and the terminal bronchiole
lumen, resulting in luminal occlusion [6, 7]. Indeed, the
Cyclosporine Nephrotoxicity, hypertension, hirsutism,
hypertrichosis, gingival hyperplasia large allograft airways develop cylindrical bronchiectasis,
Tacrolimus Neurotoxicity, hypertension, nephrotoxicity, with subsequent altered airflow, impairment of mucociliary
diabetogenic, alopecia clearance and ciliated respiratory epithelial cell loss [7].
Azathioprine Bone marrow suppression, hepatotoxicity, Potential sources of the fibroblasts responsible for the
leukopenia airway wall fibrosis and luminal obliteration include in situ
Mycophenolate Diarrhoea, nausea, vomiting, abdominal pain, proliferation of resident fibroblasts and recruitment of cir-
mofetil bone marrow suppression, hepatotoxicity, culating mesenchymal stem cells. However, it has been
leukopenia
demonstrated that bronchial epithelial cells also have the
Corticosteroids Osteoporosis, hirsutism, hypertrichosis,
ability to undergo epithelial-mesenchymal transition
diabetogenic
(EMT) mediated by transforming growth factor b1 (TGF-
b1) [8]. In the lungs, EMT has primarily been described in
Table 3 Causes of deaths occurring between 1 and 12 months after
alveolar epithelial cells and is thought to be a primary
adult lung transplantation [1] contributor to the pathogenesis of pulmonary fibrosis. A
recent study showed the direct connection between EMT
Cause of death Percentage
of patients and BOS concerning the mechanism of airway remodelling
and fibrosis, and that some immunosuppressive drugs
Acute rejection 1.8 contribute to partial EMT in bronchial epithelial cells [9].
Bronchiolitis 4.6 In fact, the only therapy for arresting functional decline of
Cardiovascular 4.5 the allograft during development of BOS is augmentation
Graft failure 17.0 of immunosuppression levels. However, in most cases, this
Infection (non-cytomegalovirus) 35.9 augmentation is ineffective when BOS is established [6].
Infection (cytomegalovirus) 2.4 Further, there is evidence that immunosuppressive
Malignancy 5.2 agents have growth-inhibitory and pro-inflammatory
Technical 3.4 effects upon human tracheobronchial epithelial cells [10].
Other 25.2 These effects may alter the integrity of the mucociliary
epithelium, causing distal airway injury and airflow
action and cough results in the continuous flow of fluid and obstruction. Indeed, the reduction in mucus-producing cells
mucus over airway surfaces. Disruption of the balance results in impairment of mucociliary clearance, potentiat-
between the ciliated epithelium, periciliary fluid and mucus ing bacterial colonization and recurrent bronchitis [10, 11].
will cause impairment of mucociliary clearance, resulting in Several in vivo and in vitro methods have been developed
microorganism proliferation, lung infection, inflammation to study the side effects of immunosuppressive drugs on
Immunosuppressant Drug Effects on Airway Clearance
airway epithelial cells and BOS development. Usually, in vivo
assays include bronchial section [12–15] and trachea [16–20]
or lung [21–24] transplantation models. Actually, heterotop-
ical tracheal transplantation in rats has been the most widely
used model, as it is an easy technique to perform and evaluate,
and because it is highly reproducible [25]. In contrast, lung
transplantation models are more appropriate for ischemia–
reperfusion and acute rejection studies, since their use to
investigate BOS presents some limitations, such as being time
consuming and technically demanding, and the high histo-
logical variability of airway lesions [6, 26]. In vitro assays are
frequently performed using human-derived tracheobronchial
epithelial cells to analyze the influence of pro-inflammatory
cytokines, growth factor and gene expression for markers of
EMT [9, 10, 27, 28]. The use of these cells in culture devoid of
other contaminating cell types is useful to examine the
response to specific immunosuppressive agents [28].
This review focuses on the current armamentarium of
immunosuppressant drugs used after lung transplantation,
their main side effects upon airway epithelial cells and the
mucociliary apparatus, and possible implications in BOS
development.
2 Immunosuppressive Drugs
2.1 Calcineurin Inhibitors
2.1.1 Cyclosporine
Cyclosporine (ciclosporin) represents a group of oligo-
peptides with 11 amino acids, and is produced as secondary
metabolites by Cylindrocarpon lucidum and Trichoderma
polysporum [29]. Cyclosporine inhibits transcription of
interleukin (IL)-2 and some other inflammatory proteins,
such as IL-3 and IL-4, tumour necrosis factor (TNF)-a and
interferon (IFN). It binds cyclophilin and prevents calci-
neurin dephosphorylation of nuclear factor of activated T
cells (NFAT), which affects activation and proliferation of
CD4? T cells through the IL-2 pathway [30]. The profound
immunosuppressive effect of cyclosporine has been dem-
onstrated in many experimental transplantation models in a
range of species, including mice, rats, guinea pigs, rabbits,
dogs, pigs and nonhuman primates [29].
Nevertheless, it is not so easy to determine the standard
dose of cyclosporine either clinically or experimentally [18].
Clinicians need to find the appropriate dose for each patient,
aiming to avoid graft rejection and to minimize toxicity.
During the first months after lung transplantation, the
cyclosporine dose is adjusted to maintain a therapeutic blood
level of around 300 ng/mL [31–34]. Experimentally, there
have been studies that considered 1.0 mg/kg/day as a low
dose and 1.5 mg/kg/day as a high dose [17]. However, this
1159
same dose (1.5 mg/kg/day) was considered low in another
study [35], which used 5.0 mg/kg/day as a high dose.
In addition, we found a study that used 5.0, 10.0 and 15.0
mg/kg/day as low doses and 25.0 mg/kg/day as a high dose
[16]. Anyway, in general, cyclosporine doses used in rat-
model transplantation studies are higher than those used
clinically, because rats have much faster hepatic metabolism
than humans, and a different body area to volume ratio [35].
2.1.1.1 In Vivo Studies BOS was originally investigated
by using a BALB/c-to-C3H trachea transplantation model,
in which grafts placed into subcutaneous tissue developed
subepithelial inflammation, epithelial necrosis and fibro-
proliferation, mimicking human BOS [36]. Using the same
model, these researchers found that only high-dose cyclo-
sporine (25 mg/kg/day), but not low doses (5, 10 or 15
mg/kg/day), significantly reduced fibroproliferation in
allografts, yet epithelial injury and cellular inflammation
still occurred [16]. Two different doses of cyclosporine (1.0
and 1.5 mg/kg/day) were tested in a heterotopic rat tracheal
allograft transplantation model, and the results showed that
both doses significantly reduced the loss of epithelium at
10 days. However, only the higher cyclosporine dose was
able to reduce the loss of tracheal epithelium at 30 days
and attenuated development of BOS [17]. Similarly, it was
shown that allografts from rats treated with cyclosporine
(5 mg/kg/day) for 2 or 4 weeks presented just a mild
degree of mononuclear cell infiltrate in the mucosal, sub-
mucosal and muscularis layers, minimal thickness of the
submucosal fibrous layer, and complete regeneration with
normal epithelial lining, which were directly related to the
absence of BOS [18]. Another study found a high inverse
correlation between the percentage epithelial coverage and
the degree of obliteration in all of the cyclosporine-treated
grafts, according to the starting day of treatment (0, 7 or
14 days after transplantation). A minimal 4 % obliteration
occurred when cyclosporine therapy (10 mg/kg daily) was
started on day 0, and the epithelium in these grafts was
predominantly columnar, ciliated respiratory cells, with
100 % coverage in all grafts [37].
This model has also been used to investigate cellular and
molecular mechanisms, as well as inhibitory effects of
cyclosporine on the BOS process. Unlike observations in
tracheal allografts excised from nonimmunosuppressed
animals, cyclosporine-treated rats (receiving 1, 2 or 5
mg/kg/day) showed decreased development of BOS in a
dose-dependent fashion, in association with downregula-
tion of epithelial major histocompatibility class II antigen
expression, IL-2R expression and infiltration of T cells.
While total epithelial necrosis and intense proliferation of
granulation tissue did occlude the airway lumen of allo-
grafts, in syngeneic tracheal grafts no such changes were
observed [38]. To test the hypothesis that the injury of the
1160
airway wall in BOS is immune mediated, another study
administered exogenous IL-2 to cyclosporine-treated rats
after heterotopic tracheal transplantation and found that
cyclosporine did not prevent obstruction of the airway
lumen. Furthermore, exogenous IL-2 administration
enhanced peritracheal lymphocyte infiltration and luminal
obstruction in cyclosporine-treated tracheal allografts [39].
A similar study in mice showed that cyclosporine failed to
block overproduction of IL-2, IFN-c, IL-4, IL-10 and
granzyme b—all related to BOS development [40].
Moreover, neutralization of IL-10 in a rat heterotopic tra-
cheal transplantation model accelerated airway oblitera-
tion, whereas administration of physiologic doses of IL-10
attenuated allograft airway fibroplasia [7].
An orthotopic tracheal transplantation model has also
been applied, with the goal of demonstrating that the
airway epithelium plays a crucial role in BOS develop-
ment. One study reported that repopulation of orthotopic
tracheal allografts with recipient-derived epithelium had a
protective effect against BOS after retransplantation of
BALB/c trachea allografts back into BALB/c mice [41].
Another study tested cyclosporine (10 mg/kg/day) in this
same model in rabbits and found neither rejection signs
nor impairment of mucociliary clearance after 10 weeks
of treatment. However, cyclosporine administration for
2 weeks or intermittently for 10 weeks was ineffective in
maintaining indefinite graft viability. Curiously, inter-
mittent pulsing of cyclosporine induced simultaneous
endothelial graft repopulation and chronic allograft
rejection [42].
In a comparative study of syngeneic and allogenic
orthotopic tracheal transplantation in mice, only nonimmu-
nosuppressed allogenic grafts presented normal ciliated
respiratory epithelial loss and impaired mucociliary func-
tion, besides a lymphocytic infiltrate [43]. Two animal
studies investigated the effects of cyclosporine (10 mg/kg),
either associated with a bronchial section or not, and reported
that cyclosporine-treated rats presented a significant reduc-
tion in mucus production from goblet cells, diminished cil-
iary beating frequency, and impairment of mucociliary
clearance at up to 90 days of therapy [12, 13]. In contrast, in
Ascaris-sensitized cats, the same dose of cyclosporine
caused goblet cell and submucosal gland hyperplasia and
hypertrophy, and inhibited airway wall remodelling [44].
Although mucociliary clearance was not measured in this
study, one can suppose that it was also impaired because of
mucus overproduction. In both situations of lesser or greater
production of mucus, diminished mucociliary clearance
facilitates retention of pathogenic microorganisms and,
consequently, respiratory infections—an important risk
factor for development of BOS [7].
Delivery of cyclosporine by aerosol in lung transplant
recipients has been studied in the last decade, both to avoid
R. Pazetti et al.
systemic toxicities of the drug and to decrease local
immunoproliferative responses and BOS. A case–control
study of lung transplant patients with biopsy-documented
BOS showed that aerosol cyclosporine in combination with
standard therapy provided a survival advantage over stan-
dard therapy alone [45]. The effects of cyclosporine on pro-
inflammatory signalling mediated by airway epithelial cells
were examined in a mouse model of acute infection by
Pseudomonas aeruginosa. The authors concluded that
cyclosporine treatment did not increase the risk of acute
pneumonia or death in the treated animals, nor did it pre-
vent an appropriate neutrophil response to infection [46].
These data were confirmed by in vitro assays.
2.1.1.2 In Vitro Studies The effects of cyclosporine upon
airway epithelial cells have also been investigated in vitro by
using either cells collected from lung transplant recipients or
human bronchial epithelial cell lines, such as BEAS-2B. One
study demonstrated that cyclosporine (10, 100, 1000 ng/mL)
exerted growth-inhibitory and pro-inflammatory effects
upon human tracheobronchial epithelial cells, which altered
the integrity of the mucociliary epithelium, facilitating distal
airway injury and airflow obstruction. Cyclosporine also
caused increased airway epithelial cell death after 5 days of
treatment. This study concluded that the reduction in mucus-
producing cells may alter mucociliary clearance, potentiat-
ing bacterial colonization and recurrent bronchitis [10].
Some years later, these researchers showed opposite effects
from inhaled cyclosporine, which decreased the secretion of
critical cytokines and chemokines from human airway epi-
thelial cells in an air–liquid-interface culture [47]. In addi-
tion, the data from another investigation provided evidence
that a high dose of cyclosporine induced production of pro-
inflammatory cytokines IL-6 and IL-8 by airway-derived
epithelial cell lines and primary epithelial cell cultures
obtained from lung brushings [27].
More recently, it was demonstrated that differentiated
airway epithelial cells collected from stable lung transplant
recipients normally secreted IL-6, IL-8 and TNF-a in
response to IL-1b stimulation. Indeed, the secretion was
not affected by treatment with cyclosporine, in contrast to
findings from studies using cells grown in submersion
culture [28]. Felton et al. [9] investigated the contribution
of cyclosporine to partial EMT in bronchial epithelial cells
and found that cyclosporine caused upregulations of the
mesenchymal markers fibronectin and vimentin. Cyclo-
sporine-treated cells also had a 5-fold increase in collagen
production and a 1-fold increase in TGF-b1 production. In
addition, cells treated with cyclosporine began to detach
from each other, losing cell–cell adhesion, and to elongate
and flatten out significantly. Different doses of cyclospor-
ine (0.1, 0.5, 1, 5 and 10 lg/mL) were tested in a human
epithelium–fibroblast interactive model to verify the role of
Immunosuppressant Drug Effects on Airway Clearance
epithelium–fibroblast interactions in the course of fibro-
proliferation, and it was found that clinically relevant
concentrations of cyclosporine (0.1–1 lg/mL) did not
affect fibroblast proliferation in monocultures. However, a
conditioned medium obtained from epithelial cells treated
with 0.1 lg/mL of cyclosporine significantly enhanced
fibroblast proliferation. These data showed that fibroblast
proliferation was stimulated by specific concentrations of
cyclosporine through mediators produced by airway epi-
thelial cells [48].
The human bronchial epithelial cell line BEAS-2B was
also used to investigate the effects of cyclosporine on the
apoptosis process, as well as on production of reactive
oxygen species and antioxidant defence. The authors found
a dose-dependent cell viability decrease, with intense
chromatin condensation and nuclear fragmentation in high-
dose cyclosporine-treated cells (100 lg/mL) [49]. They
also observed increased reactive oxygen species production
and decreased biological antioxidant potential in BEAS-2B
after cyclosporine treatment [50].
From these in vivo and in vitro studies, we can conclude
that cyclosporine promotes (or at least does not prevent)
pro-inflammatory cytokine production by airway epithelial
cells, fibroblast proliferation and impairment of mucocili-
ary clearance—all related to the BOS process. These
cyclosporine effects depend upon factors such as the dose,
the start time and the duration of treatment, which deter-
mine the success in preventing BOS development.
2.1.2 Tacrolimus
Despite having a different chemical structure from that of
cyclosporine, tacrolimus suppresses the immune system in
a similar way by inhibiting synthesis of IL-2 through
binding of a specific immunophilin, tacrolimus binding
protein (FKBP12) and, consequently, through inhibition of
the calcineurin by this complex [51, 52].
Tacrolimus is around 10-fold more potent than cyclo-
sporine [32], and the recommended starting dosage of oral
tacrolimus in adults ranges from approximately 0.1 to
0.3 mg/kg/day, but the dose may be reduced during
maintenance therapy [53]. Trough whole-blood tacrolimus
concentrations should be monitored and generally main-
tained between 10 and 20 ng/mL, depending on the time
after transplantation [30, 34]. Therapeutic blood concen-
trations of tacrolimus are usually defined as 10–14 ng/mL
during the 3-month post-lung transplantation period and
8–12 ng/mL thereafter [32, 54]. Children generally require
higher doses than adults to achieve similar whole-blood
concentrations of tacrolimus [53].
2.1.2.1 In Vivo Studies Experimentally, many studies
have investigated the effects of tacrolimus on the
1161
respiratory epithelium, aiming for greater knowledge of the
inflammatory process and BOS development after lung
transplantation.
By using the most common tracheal allograft transplan-
tation animal model, it was found that the epithelial layer was
absent in controls (receiving no treatment), while squamous
metaplasia/cuboidal epithelium was encountered in the
tacrolimus group at 14 days, but with complete regeneration
with normal epithelial lining at 28 days. In comparison with
the severe degree of mononuclear infiltrate in all sections of
the mucosal, submucosal and muscularis layers in the control
group, only a mild degree was observed in tacrolimus-treated
rats. Indeed, the thickness of the submucosal fibrous layer
was minimal (\10 lm) in the tacrolimus group but large in
the no-treatment group ([20 lm) [18].
Two studies using the same animal model confirmed
these data, showing effective preservation of tracheal
respiratory epithelium by tacrolimus in a dose-dependent
way. They found that high-dose tacrolimus (4 mg/kg)
highly significantly suppressed luminal obliteration, pre-
venting rapid epithelial destruction. A mean of 76 % of the
graft’s luminal surface was covered with epithelium, which
consisted mainly of the respiratory pseudostratified type,
and many peroidic acid-Schiff (PAS)-positive mucin-con-
taining goblet cells were found in high-dose tacrolimus
allografts. These allografts revealed even less infiltration of
mononuclear cells. Moreover, tacrolimus did not affect
smooth muscle cell proliferation [19, 20].
This same group of researchers investigated the effects of
tacrolimus administered either orally or by aerosol and found
that both routes largely prevented development of BOS and
significantly reduced epithelial injury. Indeed, they were
similarly effective in suppressing graft mononuclear infil-
tration and spot frequencies for IFN-c. However, only oral
tacrolimus sufficiently reduced IL-4-producing cells. IL-4,
IL-6 and IL-10 intragraft levels were mildly reduced by
either treatment. At 60 days, all grafts showed nearly com-
plete epithelial coverage, with a predominance of physio-
logic, ciliated, columnar epithelium [55].
More recently, these researchers investigated several
aspects of tacrolimus treatment. All animals tolerated the
tacrolimus treatment well, irrespective of the administration
route. The tracheal and bronchial epithelium in both tacrol-
imus groups was well preserved after 3 weeks. The density
of goblet cells and mucin MUC5B gene expression remained
unchanged with either route of tacrolimus treatment. No
changes in expression of zonula occludens 1 or in its distri-
bution along the cell–cell boundaries of the airway epithe-
lium and along the basement membrane were found after
tacrolimus inhalation, compared with control tracheas [56].
The effects of different doses of tacrolimus prophylaxis
and treatment on BOS development was examined in a
murine tracheal transplantation model. Tacrolimus
1162
administration was started 0, 7 or 14 days after transplan-
tation. The results showed that tacrolimus prophylaxis
dose-dependently inhibited BOS, early treatment halted
disease progression and late treatment delayed progression.
Tacrolimus prophylaxis was also associated with inhibition
of recruitment of CD4?, CD8? and IL-2R? inflammatory
cells into the allografts, suggesting a central role for IL-2 in
the development of BOS. In addition, a dose-dependent
correlation between epithelial necrosis and tracheal
occlusion was observed, suggesting that epithelial injury is
required for development of BOS. When tacrolimus treat-
ment was initiated at a time when the obliterative lesion
had already started to develop, it inhibited the progression
of BOS significantly [57].
2.1.2.2 In Vitro Studies These same effects of tacrolimus
have also been investigated in cultured cells. Different
concentrations of tacrolimus (10, 100 and 1,000 ng/mL)
were tested in human-derived tracheobronchial airway
epithelial cells. After 7 days, histology revealed no changes
in epithelial morphology or tight junction formation, and no
obvious signs of cell toxicity or cell injury were observed,
irrespective of the tacrolimus dose. Indeed, tacrolimus was
effective in preventing IFN-c, IL-10, IL-13, monocyte
chemotactic protein-1 (MCP-1) and TNF-a upregulation
after stimulation by IL-1b [56]. On the other hand, there
was evidence that tacrolimus was able to enhance produc-
tion of IL-6 and IL-8 in airway-derived cell lines and pri-
mary cells. Although high concentrations of tacrolimus
(0.1 lg/mL) enhanced IL-6 production in the epithelial cell
line A549, it did not affect IL-6 production at clinical
immunosuppressive concentrations (0.2–2.0 ng/mL) [27].
Neuringer et al. [10] showed that tacrolimus caused no
significant airway epithelial cell growth inhibition and that
IL-8 production was increased in tacrolimus-treated sam-
ples, but without statistical significance. They also reported
that the percentage multi-layered epithelium trended down
with increasing concentrations of tacrolimus (0.1 to
100 ng/mL). Another study investigated the contribution of
the bronchial EMT induced by immunosuppression and
concluded that tacrolimus-treated cells maintained epithe-
lial morphology, baseline levels of matrix protein expres-
sion and transforming growth factor production levels [9].
By using a human airway epithelial cell–lung fibroblast
interactive model, it was demonstrated that tacrolimus did
not affect fibroblast proliferation through mediators pro-
duced by the epithelial cells. Furthermore, the results
showed that the pro-proliferative effect of epithelial cell
medium on fibroblasts was not altered by pretreatment
with clinically relevant concentrations of tacrolimus
(0.001–0.01 lg/mL). These concentrations had little to no
effect on cell viability or apoptosis [11]. Subsequently,
these authors examined whether prostaglandin E2 (PGE2)
R. Pazetti et al.
inhibited primary fibroblast proliferation and whether ta-
crolimus was able to modulate PGE2 production in epi-
thelial cells. They found that basal PGE2 secretion by
epithelial cells was in the picogram range, and the highest
tacrolimus concentration (10 lg/mL) caused a significant
increase in PGE2 production. However, these data dem-
onstrated that PGE2 levels secreted by epithelial cells were
insufficient to exert an effect on fibroblast proliferation
[48].
As part of the alloreactive response, T cells induce
apoptosis of target epithelial cells by secreting the serine
protease granzyme b. On the basis of a previous study,
which reported an increase in apoptosis of epithelial cells
collected from the large airway in lung transplant recipients
[58], Hodge et al. [59] hypothesized that granzyme b would
be increased in lung transplant patients with acute rejection
and BOS, and that commonly used immunosuppressive
agents would fail to suppress this serine protease ade-
quately. Tacrolimus (25 ng/mL) was effective in reducing
granzyme b production, and this reduction was significant
for both CD4? and CD8? T cells. However, it is important
to observe that this dose was slightly higher than the
therapeutic range of 5–20 ng/mL. They concluded that
tacrolimus exerted a dose-dependent reduction in gran-
zyme b production.
Regulation of the ciliary beating frequency in the airway
cells is crucial for maintenance of mucociliary clearance
and may be dependent on frequency-modulated Ca2? sig-
nalling [5]. The frequency of ciliary beats is greater during
Ca2? oscillations than during a single Ca2? transient, and
higher frequency Ca2? oscillations serve to produce a
stable and elevated frequency of beating [60]. It was
reported that tacrolimus has the capacity to accumulate in
T lymphocytes and concentrate up to 900-fold the extra-
cellularly added concentration, which can lead to a
reduction in Ca2? accumulation and subsequently to dys-
function [61].
To elucidate the effect of tacrolimus on Ca2? oscilla-
tions in the airway epithelium, Kanoh et al. [62] investi-
gated cultured cow tracheal epithelial cells with a
Ca2? image-analysis system and showed that tacrolimus
was able to gradually attenuate and abolish the long-lasting
Ca2? oscillations induced by adenosine triphosphate in
subconfluent cells but not in confluent cells. They also
observed that tacrolimus treatment decreased the Ca2?
content in thapsigargin-sensitive stores, suggesting that the
partial depletion of the stores causes the inhibition of Ca2?
oscillations. Indeed, immunocytochemistry revealed the
existence of cytoplasmic FKBP-like immunoreactivities.
The expression of a 12-kDa FKBP was greater in sub-
confluent cells than in confluent cells, suggesting that the
12-kDa FKBP may be one of the factors that regulate Ca2?
oscillations. Therefore, tacrolimus has an inhibitory effect
Immunosuppressant Drug Effects on Airway Clearance
on the Ca2? response via intracellular FKBP, which may
result in modification of airway epithelial functions.
All of these data show that tacrolimus seems to be less
deleterious to the respiratory epithelium than cyclosporine.
Besides, it is more effective in preventing BOS develop-
ment. However, the positive effects of tacrolimus are
usually found in high dosages, which can disturb airway
mucociliary clearance.
2.2 Cell Cycle Inhibitors
2.2.1 Azathioprine
Azathioprine is a purine analogue and has a well-known
mechanism of action at the level of DNA. After its
reduction by glutathione to 6-mercaptopurine, it is enzy-
matically converted into several metabolites, which are
incorporated into replicating DNA and halt replication.
These metabolites can also block the de novo pathway of
purine synthesis by inhibiting the production of adenylic
and guanylic acid. Indeed, they can convert the co-stimu-
latory signal from CD28 into an apoptotic signal, resulting
in lymphocyte depletion [63–65].
In clinical and experimental studies, the reported typical
dose of azathioprine is in the range of 0.5–3 mg/kg/day
[14, 31, 66–69].
2.2.1.1 In Vivo Studies By using a model of bronchial
section and reanastomosis in rats, Pêgo-Fernandes et al.
[14] evaluated the effects of azathioprine (3 mg/kg/day) on
the mucociliary system for up to 30 days of treatment.
They showed that azathioprine caused only transitory
impairment of mucociliary transport in the sectioned
bronchi of rats, whereas administration of saline solution
impaired mucociliary transport for up to 30 days. In
addition, in analysis of the contact angle and frog palate
transportability of bronchial mucus samples, they observed
that azathioprine preserved mucus viscoelastic properties
in comparison with saline treatment.
2.2.1.2 In Vitro Studies Azzola et al. [11] investigated
the in vitro effect of azathioprine (0.01–50 mg/L) on the
proliferation capacity of primary human lung fibroblasts
obtained from transbronchial biopsies of lung transplant
recipients. There was no effect of azathioprine on fibroblast
proliferation up to a concentration of 1 mg/L. Azathioprine
showed relevant antiproliferative effects only at higher
doses, which are contraindicated in vivo because of side
effects.
On the basis of knowledge of EMT in renal tubular
epithelial cells induced by transplant immunosuppressive
therapy, a recent and very well-conducted study investi-
gated whether azathioprine contributed to EMT in airway
1163
epithelial cells. The results showed that azathioprine-trea-
ted cells presented irregularity in cell shape without the
loss of cell–cell contact. The epithelial marker aquaporin 5
was highly expressed in vehicle-treated cells, but in the
presence of azathioprine, it nearly disappeared. In contrast,
the expression of tight junction protein E-cadherin was not
reduced in azathioprine-treated cells, maintaining cell–cell
junctions and localization of E-cadherin at the cell mem-
brane. The mesenchymal markers fibronectin and vimentin,
which are normally expressed at low or undetectable levels,
showed slight and a moderate increases, respectively, with
azathioprine treatment. Cells treated with azathioprine
generally maintained cytoskeletal structure and integrity of
the actin ring. Indeed, azathioprine-treated cells showed
significant nuclear blebbing, typically associated with
preapoptotic cells, and increased collagen production
ranging from 3- to 4-fold [9].
Azathioprine is one of the oldest immunosuppressive
drugs used after lung transplantation. However, the literature
is poor with regard to studies reporting its isolated and direct
side effects on airway epithelial cells, because it is com-
monly used as part of a standard triple-drug therapy. Some
clinical trials have compared azathioprine with mycophen-
olate [70, 71], sirolimus [72] or everolimus [73] in cyclo-
sporine- or tacrolimus-based therapies. All of them found
similar incidences of infection and acute rejection episodes,
as well as BOS, at 1 and 3 years after transplantation.
It is not possible to draw firm conclusions from these few
experimental and clinical studies, but it seems that azathi-
oprine does not cause important damage in airway epithelial
cells, which could be related to BOS development.
2.2.2 Mycophenolate Mofetil, Mycophenolate Sodium
and Mycophenolic Acid
Immunosuppression by mycophenolate occurs through its
active compound, mycophenolic acid, a selective and
reversible inhibitor of inosine monophosphate dehydroge-
nase. Mycophenolate impairs the rate-limiting enzyme in
de novo synthesis of guanine nucleotides. T and B cells are
more dependent on this pathway than other cells are [74,
75]. Other potential mechanisms of immunosuppression
include inducing apoptosis of activated T cells; decreasing
expression of adhesion molecules, resulting in decreased
recruitment of inflammatory cells; and decreasing induc-
ible nitric oxide production and resultant tissue damage
[76].
The standard dose of mycophenolate depends upon both
its formulation and its combination with calcineurin
inhibitors. In general, the dose used with cyclosporine is
3 g/day, whereas in combination with tacrolimus it is 2 g/day
[31, 74, 76]. Experimentally, the dose used in vivo ranges
from 10 to 40 mg/kg/day [15, 18, 37, 38].
1164
2.2.2.1 In Vivo Studies The role of mycophenolate in the
BOS process was examined by using a reproducible model
of heterotopically transplanted rat tracheas. A mean oblit-
eration rate of 47 % was observed after treatment with
mycophenolate (40 mg/kg/day) from day 0 to day 27.
Indeed, the epithelium was flattened, cuboidal, sporadic
and interrupted in coverage, and was only seen lining tra-
cheas with minimal obliteration. The result was worse
when treatment was started on day 7—the obliteration
value reached 93 %, and no epithelium was present [37].
Similar results were achieved by using the same model,
where mycophenolate at a dose of 20 mg/kg/day did not
inhibit development of BOS. In this group, respiratory
epithelium had disappeared from every graft, and the tra-
cheal lumen was occluded 30 days after transplantation.
Mycophenolate slightly reduced inflammatory cell IL-2R
expression at peak inflammation but did not alter the
intensity or cell constitution of the inflammation. In addi-
tion, mycophenolate at a dose of 40 mg/kg/day resulted in
the deaths of three of four animals, due to anaemia and
weight loss within 2 weeks, and in the one surviving ani-
mal, the graft was totally occluded by intense granulation
tissue [38]. These data were corroborated by Yonan et al.
[18], using the same animal model. After mycophenolate
treatment (40 mg/kg/day), the epithelial layer was absent
in trachea allografts, and there was a moderate degree of
mononuclear cell infiltrate in all sections of the mucosal,
submucosal and muscularis layers in the mycophenolate
groups. Indeed, the thickness of the submucosal fibrous
layer was intermediate in the mycophenolate groups
(10–15 lm) and large in the no-treatment group ([20 lm).
The effects of mycophenolate upon mucociliary clear-
ance were tested in a bronchial section model in rats by
measuring in situ ciliary beat frequency, mucociliary
transport velocity and in vitro mucus transportability. Data
collected from the rat mucus samples tested in the frog
palate model showed that mycophenolate did not alter any
transportability property for up to 30 days of treatment
after surgery. However, mycophenolate-treated rats had a
significant decrease in ciliary beat frequency values from
sectioned bronchi on postoperative day 30. Mucociliary
transport velocity was significantly slower in the myco-
phenolate group than in the saline-treated group. The
conclusion was that this impairment of mucociliary clear-
ance could be related to the high incidence of respiratory
infections in transplant recipients [15].
2.2.2.2 In Vitro Studies The effects of mycophenolate
upon epithelial cells have also been investigated in dif-
ferent cultured cell lines. For example, mycophenolate
demonstrated a biphase effect on conjunctival goblet cell
proliferation and upregulation of mucin-5AC, a high-
molecular-weight glycoprotein and the major component
R. Pazetti et al.
of the mucus layer [77]. Interestingly, an enhancing effect
of mycophenolate on proliferation of conjunctival goblet
cells at lower concentrations was demonstrated in this
study. At a dose of 0.25–1 ng/mL, mycophenolate
increased cell viability up to 20.6 %. However, a dose of
mycophenolate 100 ng/mL for 24 h reduced cell viability
by 49.7 %. This finding is consistent with studies testing
mycophenolate (5–50 ng/mL) on lymphocytes, monocytes,
vascular smooth muscle cells, fibroblasts and human
intrahepatic biliary epithelial cells [78, 79]. Despite poor
knowledge of the genes associated with MUC5AC pro-
duction in conjunctival goblet cells, it was demonstrated
that SAM-pointed domain-containing Ets-like factor
(SPDEF) is the key genetic switch in pulmonary goblet
cells. SPDEF induced messenger RNA (mRNA) expression
of MUC5AC in progenitors of goblet cells, with no asso-
ciated proliferation of goblet cells in the respiratory tract
[80]. This result is similar to a mismatch between
MUC5AC mRNA expression and proliferation of the
associated conjunctival goblet cells under treatment with
higher concentrations of mycophenolate, indicating that
increased secretion of mucin in tear film could be due to
enhanced function of the individual conjunctival goblet
cells rather than increased numbers of the cells [77].
The role of mycophenolate in EMT was studied in the
bronchial epithelial cell line RL-65. The results revealed that
cells treated with mycophenolate demonstrated significant
loss of aquaporin 5 expression, whereas expression of
E-cadherin remained consistent. Indeed, mycophenolate
elicited increased expression levels of fibronectin. Myco-
phenolate treatment resulted in loss of the actin ring and
formation of visible stress fibres. Mycophenolate also
resulted in loss of E-cadherin localization at the cell mem-
brane and a 3-fold increase in collagen production. Myco-
phenolate-treated cells begin to detach from each other,
losing cell–cell adhesion, and to elongate and flatten out
significantly, adopting more fibroblast-like morphology [9].
Nevertheless, by using a primary culture of human lung
fibroblasts obtained from lung transplant recipients, it was
shown that mycophenolate (0.001–5 mg/L) had a potent
antifibroproliferative effect at concentrations achieved
clinically. Mycophenolate at a dose of 0.5 mg/L inhibited
50 % of cell proliferation, which was completely stopped
when mycophenolate was given at concentrations close to
clinical trough levels (1 mg/L) [11]. It was reported that
mycophenolate exerts dose-dependent inhibition of guano-
sine-dependent inducible nitric oxide synthase expression
and nitric oxide production in fibroblasts [81]. Thus, it is
possible that the antifibroproliferative effect of mycophen-
olate is due to inducible nitric oxide synthase inhibition.
As was mentioned previously, pro-inflammatory cyto-
kines are related to impairment of respiratory epithelium
and have been studied extensively. Borger et al. [27]
Immunosuppressant Drug Effects on Airway Clearance
demonstrated that addition of mycophenolate (C0.1 lg/
mL) to A549 cells enhanced production of IL-6 and IL-8
proteins by airway-derived cell lines and primary cells. On
the other hand, Allison and Eugui [78] reviewed many
studies and found that 48 h after stimulation, mycophen-
olate inhibited production of all cytokines that were stud-
ied, including IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-c
and TNF-a. They also observed that in clinically attainable
concentrations (1–10 lM), mycophenolate suppressed
proliferation of arterial smooth muscle cells and fibroblasts,
which may be relevant to prevention of BOS.
In summary, some studies observed that mycophenolate
was effective in preventing fibroblast proliferation. How-
ever, the majority of these studies showed that myco-
phenolate caused deleterious effects on epithelial tissue and
mucociliary clearance.
2.3 Corticosteroids
Prednisone, prednisolone and methylprednisolone are the
most common corticosteroids used for induction, mainte-
nance and anti-rejection therapy in lung transplantation
[74]. The immunosuppressive action of corticosteroids is
complex and occurs by several mechanisms including (i) T
lymphocyte depletion; (ii) inhibition of T cell activation
and proliferation; and (iii) inhibition of monocyte chemo-
taxis and migration to sites of inflammation [65].
Most physicians have adopted the use of a moderate
dose of steroids in the early postoperative period, aiming to
avoid adverse effects on bronchial healing. Methylpred-
nisolone is commonly employed at a dose of 0.5 mg/kg
intravenously twice daily for 3 days postoperatively before
initiation of an oral dose of prednisone 0.5 mg/kg/day for
the first 3 months, which is then tapered to 15 mg/day by
6 months and finally to 15 mg on alternate days by 1 year
[31, 74, 82].
2.3.1 In Vivo Studies
Since airway inflammation has been considered an
important feature in established BOS, a high dose of
inhaled steroid was tested as adjunctive standard immu-
nosuppressive treatment in a randomized, double-blind
study of 30 stable lung transplant patients, but no effect on
BOS or survival was seen [83]. Steroid effects on airway
tissue have commonly been investigated in patients with
asthma or chronic obstructive pulmonary disease because
of the histological similarities between BOS and these
pathological conditions. The effects of inhaled corticoste-
roids (budesonide 0.25–0.50 mg once daily) and systemic
corticosteroids (oral prednisone 40 mg once daily) on
mucociliary clearance were tested in outpatient asthma
through an in vivo, randomized, placebo-controlled, single-
1165
blind study. The results showed that treatment with
budesonide did not affect mucociliary clearance. However,
treatment with prednisone was associated with a significant
improvement in mucociliary clearance at 24 h [84].
The airway epithelium is a primary target of the anti-
inflammatory actions of inhaled steroids, which restore
epithelial integrity by suppressing the expression of mul-
tiple epithelial-derived cytokines and chemoattractants, as
well as by promoting the maintenance of proper cell–cell
adhesion and decreasing mucus hypersecretion [85].
Investigating the route of administration of steroids, it was
found that oral prednisolone treatment given to stable
asthmatics led to a significant improvement in clearance
from the peripheral and intermediate regions of the lungs,
but clearance from the inner region of the lungs was sig-
nificantly increased only in six patients who coughed rel-
atively infrequently prior to treatment [86].
Although the development of topical glucocorticoids
drastically reduced the adverse effects of systemic gluco-
corticoids, localized adverse effects are most commonly
encountered with the use of inhaled steroids. Hanania et al.
[87] critically reviewed this evidence but found no histo-
logical damage to the tracheobronchial epithelium or
connective tissues after long-term use of inhaled steroids.
In addition, they did not observe any reported changes in
exfoliative cytology. They compared this finding with the
common dermal and connective tissue atrophy seen in the
skin after topical application of the same drugs, and
hypothesized that this difference presumably reflects the
relatively low concentration of inhaled steroid per unit
surface area in the lung, its relatively rapid clearance from
the airways by systemic absorption, and possible differ-
ences in lung fibroblast sensitivity to glucocorticoids.
The effects of prednisone on the mucociliary apparatus
of rats was tested experimentally. The results showed that
prednisone reduced mucus transportability (0.65–2.50
mg/kg/day) but impaired mucociliary clearance only after a
high dose (2.5 mg) [88]. Indeed, a standard dose (1.25 mg)
improved mucus transportability in the animals undergoing
bronchial section and reanastomosis up to 30 days after
surgery [89].
2.3.2 In Vitro Studies
It was demonstrated that prednisolone had a differential
effect on IL-6 and IL-8 production by airway-derived cell
lines and primary cells. At low concentrations (0.001 and
0.01 lg/mL), prednisolone significantly enhanced cytokine
production. However, the opposite effect was observed at
high concentrations (0.1–10 lg/mL). Prednisolone was
also able to repress the binding activity of nuclear factor
kappa B (NF-jB) and activator protein-1 (AP-1) in
the epithelial j cell line BEAS-2B. Since both NF-jB and
1166
AP-1 are implicated in the regulation of IL-6 and IL-8 gene
transcription in epithelial cells, the conclusion was that
repression of the binding activities of these transcription
factors may well explain the reduced production of IL-6
and IL-8 [27].
Doerner and Zuraw [90] investigated whether TGF-b1
stimulates primary human bronchial epithelial cells to
undergo transition to a mesenchymal phenotype, and whe-
ther this transition can be abrogated by corticosteroid
treatment. Treatment with TGF-b1 significantly reduced the
expression level of the epithelial adherence junction protein
E-cadherin and markedly induced mesenchymal marker
proteins such as collagen I, tenascin C, fibronectin and
a-smooth muscle actin mRNA in a dose-dependent manner.
The process of mesenchymal transition was accompanied
by a morphological change towards a more spindle-shaped
fibroblast cell type with a more motile and invasive phe-
notype. Corticosteroid pretreatment did not significantly
alter the TGF-b1 induced transition. Since asthma has been
strongly associated with increased expression of TGF-b1 in
the airway, they concluded that EMT may contribute to the
contractile and fibrotic remodelling process.
A similar study showed that prednisone-treated cells
exhibited a typical epithelial morphology, including a
consistent round shape and a tightly junctioned monolayer,
nearly indistinguishable from untreated control cells.
Overall expression of E-cadherin was not reduced in the
presence of prednisone. However, aquaporin 5 expression
nearly disappeared. The mesenchymal markers fibronectin
and vimentin were normally expressed at low or unde-
tectable levels. Prednisone-treated cells maintained cell–
cell junctions and localization of E-cadherin at the cell
membrane. Indeed, they did not exhibit a significant
increase in soluble collagen production. They also main-
tained cytoskeletal structure and integrity of the actin ring
at the membrane of each cell [9].
The beneficial effects of corticoids in controlling epi-
thelial remodelling and promoting the wound-healing
process have been also observed in nasal polyposis. Li
et al. [91] studied the role of p63, a member of the p53
family, in the mechanism of epithelial remodelling in
patients with nasal polyposis and its response to corticoid
therapy. They found that oral prednisone (10 mg three
times daily for 10 days) could relieve the abnormality of
nasal polyposis epithelium and reduce ectopic p63
expression. The results also showed that p63-positive cells
in both nasal polyposis and control epithelium were only
detected in the basal cell layers. Moreover, in patients with
nasal polyposis, a significant increase in the number of
p63-positive basal cells was confirmed. together with
increasing levels of p63 mRNA. Moreover, the overex-
pression of p63 was correlated with epithelial hyperplasia
in patients with nasal polyposis. They concluded that since
R. Pazetti et al.
p63 controls the transcriptional activity of a variety of
downstream genes, such as the epithelial cell cycle and cell
growth, proliferation, differentiation and death, it not only
represents an extraordinarily specific biomarker for airway
basal cells but also regulates the self-renewal of basal cells
and would contribute to development of epithelial remod-
elling in chronically inflamed airway mucosa, such as that
of patients with nasal polyposis.
It seems that inhaled and systemic steroids do not cause
significant impairment of airway epithelial tissue. How-
ever, they are not able to prevent BOS development.
3 Conclusion
The present review highlights the influence of most com-
mon immunosuppressive agents that are used after lung
transplantation on airway epithelial tissue. In general, the
studies showed that the negative side effects of these drugs
are dose dependent. However, cyclosporine and myco-
phenolate presented a worse impact on respiratory epithe-
lium. Currently, all immunotherapy regimens appear to be
roughly equivalent in terms of prevention of rejection, and
decisions regarding selection should be based mainly on
side effect profiles and efficacy in individual patients.
Indeed, the potency of the agents now available is such that
the challenge is to avoid overimmunosuppression and the
problems of infection and malignancy that accompany
excessive non-specific immunosuppression. After review-
ing the literature, we could ask whether the finding of a
significant effect of medication on mucociliary clearance is
of clinical relevance. However, further research is needed
to more precisely establish the clinical benefit of a specific
agent in patients and to examine the exact relationship
between alterations in mucus transport induced by a certain
drug and its clinical impact on patients.
Acknowledgments No funding was used in the preparation of this
review. The authors have no conflicts of interest that are directly
relevant to the content of the review.
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