1 Institut National de la Santé et de la Recherche Médicale U506, 94807 Villejuif, France; 2 Cystic Fibrosis Research Laboratory, and 3 Herzenberg Laboratory, Stanford University, Stanford, California 94305
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ABSTRACT |
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Most cystic fibrosis (CF) patients die of lung failure, due to the combined effects of bacterial infection, neutrophil-mediated inflammation, and airway obstruction by hyperviscous mucus. To this day, it remains unclear where and how this pathological vicious circle is initiated in vivo. In particular, it has proven difficult to investigate whether inflammatory pathways are dysregulated in CF airways independently of infection. Also, the relative involvement of large (tracheobronchial) vs. small (bronchiolar) airways in CF pathophysiology is still unclear. To help address these issues, we used an in vivo model based on the maturation of human fetal CF and non-CF small airways in severe combined immunodeficiency mice. We show that uninfected mature CF small airway grafts, but not matched non-CF controls, undergo time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clear-cut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection.
severe combined immunodeficiency; neutrophils; bronchioles
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein and affects the normal function of the respiratory, digestive, genital, and skin tracts. Lung disease, which associates airway obstruction, infection, and inflammation, is responsible for most of the morbidity and mortality in CF (6) and, therefore, constitutes a major focus for research. The prevailing view at present is that CF lung inflammation is a secondary response (1), possibly exacerbated (14), to infections, with the infections themselves resulting from abnormal secretions. Therefore, most experimental studies on CF lung disease have investigated the role of gland-containing large airways (trachea, bronchi), from which supposedly abnormal secretions originate (27). Another consequence of this theoretical framework is that, therapeutically, mucolytics and antibiotics are more commonly prescribed than anti-inflammatory drugs in the treatment of CF lung disease (15).
Importantly, CFTR is also expressed at significant levels in distal areas of the lung, both in small airways (bronchioles) and areas of gas exchange (alveoli) (7). Within CF bronchioles, inflammation is detected very early on, featuring massive influx of neutrophils into the lumen, sometimes in the absence of detectable infection (3, 13, 17). In contrast, alveoli appear grossly preserved throughout the course of CF lung disease, except for air trapping (due to obstruction of upstream conducting airways) and end-stage emphysema. Owing to the lack of relevant animal models and to existing ethical and practical hurdles when it comes to studying small airway physiology in living patients, it has been very difficult so far to investigate the mechanisms of CF small airway disease.
In particular, it remains unclear which function(s) CFTR mediates in small airways and whether its absence results in a proinflammatory phenotype, independently of infection. Another long-lasting mystery lies in the fact that, despite robust CFTR expression in the fetal lung (9, 25), CF lungs are not grossly impaired before birth as demonstrated by histopathological studies of 1) lungs from CF fetuses and infants who died of meconium ileus (5, 19) and 2) developing lungs from cftr knockout mice (10). It is all the more surprising that other CFTR-expressing organs, such as the vas deferens, the pancreas, or the intestine, can be severely impaired during gestation (6). One possibility is that the main role of CFTR in distal airways is to contribute to fluid absorption (8), which is of crucial importance at and after birth only. If this is true, CFTR would have to exert only redundant, if any, function during gestation, hence the lack of lung manifestations of CF in utero.
In previous work, we introduced human fetal tracheobronchial grafts in severe combined immunodeficient (SCID) mice as a surrogate model for mature human large airways (2, 16, 22, 23). This model has the important advantage of relying on human tissues that have never been infected, thus allowing one to investigate primary defects linked to CFTR mutations. In mature human CF large airway grafts, we observed primary alterations of the immunoinflammatory balance [increased intraluminal interleukin (IL)-8 secretion and mislocalization of subepithelial human and mouse leukocytes] before any infection (22). Yet, infectious stimuli were necessary to provoke intraluminal leukocyte infiltration and to inflict significant tissue damage to the mucosa of CF large airway grafts, compared with that of non-CF controls.
To extend our results obtained on large airway grafts, we sought, in the present study, to investigate the inflammatory status of CF and non-CF human fetal lung grafts consisting of bronchiolar (small airways) and alveolar tissue. We show that CF lung grafts, and not non-CF controls, develop progressive intraluminal inflammation mediated by host neutrophils, eventually leading to the destruction of the lung parenchyma, before any infection.
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METHODS |
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Human fetal tissues and graft confection.
Fetal tissues were obtained after elective or medically indicated
termination of pregnancy in compliance with current French legislation.
Work was approved by the Ethics Committee for Life Sciences of the
Centre National de la Recherche Scientifique (CNRS). Details of
gestational age and engraftment time of the grafts used in the study
are presented in Table 1. CF and non-CF
fetal lung rudiments were dissected into 1-cm3 pieces of
lung tissue, including both bronchiolar and alveolar regions. For each
fetal lung rudiment, two pieces were kept as controls before
engraftment and processed for immunohistological analysis as described
below. Upon availability, from one to four other lung tissue pieces per
fetal lung rudiment were implanted subcutaneously in SCID mice, as
described previously (16). Grafts originating from a given
rudiment (either CF or non-CF) were implanted in mice from at least two
litters and harvested sequentially over time. Littermates were
implanted with both CF and non-CF grafts to reduce the risk of
host-derived differences. In total, 22 non-CF grafts from 10 independent tissues and 21 CF grafts from 9 independent tissues were
analyzed. Mice were maintained in strict axenic conditions in our
animal facility, and each was anesthetized with an intraperitoneal injection of 0.4 ml of etomidate (Hypnomidate; Janssen-Cilag) before
any manipulation.
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Immunohistochemistry. At indicated times (see Table 1), grafts were harvested from the host, fixed in 10% formaldehyde in phosphate-buffered saline solution, frozen, and cut into serial 5-µm-thick sections as previously described (22). Serial sections were stained with either 1) biotinylated mouse isotype control antibodies or biotinylated mouse primary antibodies against various human antigens (see below) or 2) biotinylated rat isotype control antibodies or biotinylated rat primary antibodies against various mouse antigens (see below), followed by fluorescent red streptavidin-Cy3 (DuPont-NEN, Les Ulis, France). Human markers tested were CD3 (pan-T cell marker; BD Pharmingen, Le Pont du Claix, France), CD15 (granulocytes; Dako, Trappes, France), CD20 (B cells; Immunotech, Marseilles, France), CD31 (endothelial cells; Dako), CD38 (plasma cells; BD Pharmingen), CD45 (pan-leukocyte marker, Dako), CD56 (NK cells; Immunotech), CD68 (macrophages; Dako), glycophorin A (Gly A, erythrocytes; Dako) and tryptase (mast cells; Dako). Mouse markers were Gr1 (neutrophils; BD Pharmingen), Ly5 (pan-leukocytes; BD Pharmingen), and Mac1 (activated leukocytes; BD Pharmingen). Sections were counterstained with Gill's hematoxylin. Control pieces of dissected CF and non-CF lung tissues saved before engraftment were processed identically to grafts.
Data collection and analysis.
For each donor tissue, two independent pieces were sectioned, and
serial sections were used as follows: 1) 20 sections from each (spanning the entirety of the piece) were stained with anti-CD45 (to detect all human leukocytes) and 2) 10 sections were
stained with each of the other antibodies listed above. For each graft, serial sections were used as follows: 1) 40 sections
(spanning the entirety of the graft) were stained with anti-CD45,
2) 40 sections were stained with anti-Ly5 (to detect all
murine leukocytes), and 3) 10 sections were stained with
each of the other antibodies listed above. Sections were screened using
an epifluorescence microscope (Nikon, Champigny-sur-Marne, France) by
an observer (R. Tirouvanziam) blinded to the gestational age,
engraftment time, and genotype of tissues. Individual sections were
exhaustively scanned at low magnification (×10). The bright
fluorescent signal generated by our amplified staining method (primary
antibody, biotinylated secondary antibody, streptavidin-Cy3) allowed us to successfully identify even isolated single positive events at this
low magnification. After results from all sections were compiled, each
control tissue and graft were rated qualitatively for the presence or
absence of the given markers in the mesenchyme, epithelium, and/or
lumen. Observations at higher magnification (×50 and ×250) were
occasionally performed to confirm low magnification results, but no
systematic attempt was made at quantifying the expression of each of
the markers. Finally, to provide a general index of leukocyte origin
and localization, we attributed to each control tissue and graft two
nominal scores reflecting the presence of human (based on anti-CD45
staining, uppercase letters) and murine (based on anti-Ly5 staining,
lowercase letters) leukocytes in the following compartments: mesenchyme
(M/m), epithelium (E/e), lumen (L/l), or none (O/o). Combined scores
were given when leukocytes were present in several compartments, e.g.,
"m-l" for murine cells being present in both the mesenchyme and the
lumen. Correlations were assessed using the Logistic fit function of
JMP software (SAS Institute, Cary, NC) and considered statistically
significant in the likelihood ratio 2-test with
P < 0.01. Differences between means were assessed by t-test, with P < 0.01 considered significant.
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RESULTS |
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No difference is observed between CF and non-CF fetal small airways
before engraftment.
CF and non-CF fetal lung rudiments obtained ranged from 8 to 16 wk of
gestation (Table 1); the gestational age was not different between the
two groups (11.3 ± 1.0 vs. 11.4 ± 0.7 wk, P > 0.45, t-test). These rudiments showed
histological patterns typical of pseudoglandular to early canalicular
stages of lung development. The presence of human leukocytes in control
tissues (Table 2) was highly correlated
with gestational age (P < 0.0001, likelihood ratio
2-test) but not with genotype (P > 0.88). More precisely, we observed that past 11-12 wk of
gestation, both CF and non-CF fetal lung rudiments were colonized by
scattered CD45+ leukocytes, mostly tryptase+
mastocytes, located in the mesenchyme. Scattered mesenchymal macrophages were detected from 13 wk on, whereas luminal macrophages were mostly visible in the oldest tissues (around 16 wk of gestational age). CD3+ T cells, CD15+ granulocytes,
CD20+ B cells, CD38+ plasma cells, and
CD56+ NK cells were absent from both CF and non-CF tissues.
No human leukocytes were found in the epithelium of either CF or non-CF control tissues. The mucosa of bronchiolar regions in all control tissues and the septae of emerging alveolar areas in the oldest fetal
rudiments showed intense staining with CD31, demonstrating the presence
of an extensive endothelial network, within which Gly A+
human erythrocytes were detected. Consistent with previous data showing
the absence of gross pathological alterations in CF lungs before birth
(5, 10, 19), we found no difference between CF and non-CF
lung rudiments. In particular, there was no sign of mesenchymal,
epithelial, or intraluminal inflammation in CF rudiments compared with
non-CF. As expected, all control tissues were negative for murine
antigens.
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Human endothelium and erythrocytes disappear and resident human
leukocyte populations are restricted to mast cells in both CF and
non-CF grafts.
All grafts underwent considerable macroscopic growth in the SCID host,
reaching a final volume of up to 5 cm3. After 6-8 wk
in the host and regardless of their gestational age, all grafts
featured mature bronchioles and alveoli as previously reported
(16). CD31+ and Gly A+ cells were
undetectable by 5 wk after engraftment, demonstrating a rapid loss of
human endothelial cells and of human erythrocytes, respectively, on
engraftment into the mouse host. When screened for human leukocyte
antigens (Table 2), grafts were either negative (O category) or
positive in the mesenchyme only (M category). No graft showed the
presence of intraepithelial and/or intraluminal human leukocytes. The
presence of human leukocytes in the mesenchyme of the grafts was highly
dependent on gestational age (P < 0.0001, likelihood
2 ratio analysis) but independent of engraftment time
and genotype (P > 0.05 for both). Indeed, grafts below
11-12 wk of gestational age had not been colonized by human
leukocytes at the time of implantation and remained devoid of such
cells once engrafted in the host. In both CF and non-CF grafts with
initial gestational age >11-12 wk, tryptase-positive human
mastocytes were maintained in the mesenchyme. No CD68+
macrophages were found in either the mesenchyme or the lumen of the
grafts, even those originating from fetal tissue that contained resident macrophages at the time of implantation.
Murine leukocytes migrate in the mesenchyme of all grafts.
We next examined the presence of host-derived cells in human fetal lung
grafts. Concomitant with the involution of human capillaries and
erythrocytes, murine capillaries (human CD31, not shown)
developed in the bronchiolar mucosa and interalveolar septae, carrying
mouse erythrocytes (human Gly A
, not shown). This
confirms our previous observations that both CF and non-CF lung grafts
are vascularized both externally and internally by host-derived
endothelium (16). Blinded immunohistochemical analysis
further revealed that all grafts contained a population of host-derived
Ly5+ leukocytes in the mesenchymal compartment (Fig.
1). This is in agreement with our
previous studies in proximal grafts showing that murine leukocyte
migration occurs in the mesenchyme of both CF and non-CF grafts
(22, 24).
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Murine leukocytes specifically invade the lumen of long-term CF
grafts.
However, blinded analysis also revealed that, besides being present in
the bronchiolar and alveolar mesenchyme of all grafts, Ly5+
murine leukocytes were also present in the lumen of a subgroup of
grafts (m-l category). We found the intraluminal presence of murine
leukocytes to be linked with very high significance to both the CF
genotype (P < 0.0001, Fig. 1A) and to
prolonged engraftment time (P = 0.0057, Fig.
1C) but not to gestational age (P > 0.47, Fig. 1B). Intraluminal infiltration with murine leukocytes
was not dependent on whether the grafts contained human leukocytes or
not (P > 0.13, Fig. 1D). Interestingly,
none of the grafts, neither non-CF or CF, scored positive for the
presence of murine leukocytes in the epithelium. In non-CF grafts,
Ly5+ murine leukocytes were consistently restricted to the
mesenchyme (Fig. 2, A and
B). In CF grafts with intraluminal infiltration of host
leukocytes, neutrophils represented an important intraluminal subpopulation (Fig. 2, C and D). Further
qualitative differences existed, with some CF grafts showing loose
infiltration of some but not all luminal areas (Fig. 2, C
and D), whereas others had their lumen packed full of
leukocytes (Fig. 2, E and F). Overall, we
observed mouse inflammatory cells in the lumen of 11 of 19 CF grafts
but in 0 of 22 non-CF grafts. Among groups of CF and non-CF grafts with
an engraftment time 15 wk, 11 out of 12 (92%) CF grafts showed
intraluminal inflammation, whereas 0 of 12 (0%) non-CF grafts showed
signs of intraluminal inflammation. These groups were matched for
gestational age and engraftment time [gestational age (in wk):
11.4 ± 0.7 (CF, range: 8-16) vs. 12.0 ± 1.0 (non-CF, range: 7.5-16), P > 0.31; engraftment time (in
wk): 31.3 ± 5.2 (CF, range: 15-65) vs. 26.1 ± 4.5 (non-CF, range: 15-65), P > 0.22], therefore
pointing out to the CF genotype as a decisive factor in triggering
intraluminal inflammation. Within the CF group, intraluminal
inflammation is associated with prolonged engraftment time (Fig.
1E).
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DISCUSSION |
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We have developed human fetal CF lung rudiments to full maturation, as grafts in the SCID mouse. We show that, independently of any infection, long-term CF lung grafts, but not non-CF controls, undergo massive intraluminal mouse neutrophil infiltration and progressive tissue destruction. This result provides strong evidence that CF lung inflammation can arise directly from CFTR mutations.
In the fetal pseudoglandular and early canalicular lung tissues analyzed before engraftment, mast cells and macrophages are the only resident leukocyte subsets detected. Mast cells reside in the parenchyma, whereas macrophages are in the parenchyma and in the lumen. During in utero lung development, two distinct waves of macrophage colonization occur (18). The first wave involves parenchymal macrophages, which have been proposed to play an active role in branching morphogenesis and to involute thereafter. The second wave involves cells that migrate from the blood into the alveoli from the late canalicular stage on. Neither of these two populations is found in mature grafts. The disappearance of the first subset is consistent with the fully mature state of grafts and hence with the probable arrest of the branching process. The absence of the second subset reflects the inability of the alveolar macrophages present at the time of implantation in the oldest tissues (~16 wk of gestational age) to survive and proliferate in the grafts. In contrast, mast cells are maintained, even in the long term, within the grafts. This may point to a particular capacity of mast cells to survive (and possibly proliferate) in the lung parenchymal microenvironment. We found no correlation between the presence and localization of human leukocytes and either the genotype (CF or non-CF) or the engraftment time of the grafts.
Irrespective of the presence of human leukocytes, we have observed that some CF grafts undergo spontaneous intraluminal infiltration with host leukocytes. This phenomenon occurs independently of exposure to air (as grafts are developed subcutaneously in the host) and independently of infection (as tissues are of fetal origin and are maintained in axenic conditions through the experimental process). Moreover, it occurs independently of the initial gestational age of the grafts but appears closely linked to a prolonged engraftment time, because it is observed only in CF grafts maintained past 15 wk. To date, this is the first feature of mature grafts (either proximal or distal) that we find associated with an increase in engraftment time. Indeed, we showed previously that both similarities [in histology (16), proximal airway fluid rheology and composition (2)] or dissimilarities [in ion transport properties (23) or proximal airway fluid IL-8 levels (22)] between CF and non-CF mature grafts were independent of engraftment time.
To account for these observations, we propose that, since distal grafts mature and are maintained in the SCID host as closed sacs, either an excess of proinflammatory factors or a deficiency of anti-inflammatory factors secreted by the CF epithelium leads, with time, to an accumulating balance of cross-specific proinflammatory signals that recruit host neutrophils to the grafts' lumen. In general, intraluminal infiltration of inflammatory cells can be very deleterious to epithelia and, for that reason, is subject to strong regulation. Hence, the intraluminal leukocyte infiltration of long-term CF distal grafts, but not of non-CF controls, points to a major alteration in leukocyte containment, which, once initiated, might function as a vicious inflammatory circle such as that described in CF lungs in vivo (14).
We propose that our enclosed model magnifies a phenomenon present in CF distal airways in utero but masked by the constant flushing of the open lung lumen. By allowing for effective concentrations to be reached with prolonged engraftment time, our closed model probably unmasks a CF-related alteration in leukocyte recruitment that has previously gone unnoticed in histopathological studies of open lungs of developing CF mice (10) and, more importantly, of human CF fetuses and infants (5, 19). We further propose that after birth, in vivo, any condition that reduces clearance or further stimulates inflammation (like local, low-level infections) may amplify the primary proinflammatory defect identified in our study.
Recently, Hubeau et al. (11) found increased macrophage numbers in late-stage CF lungs developed in utero, compared with controls. Although this phenomenon could not be reproduced here [since we establish grafts from early-stage fetal tissues (<16 wk), which are not yet subject to intralveolar macrophage migration], it brings forth further evidence that CF lungs in utero display a basal alteration in the recruitment of inflammatory cells. Several mechanisms accounting for such an alteration have been proposed in CF, involving both upstream events [e.g., abnormal transcriptional regulation of genes involved in inflammation (12, 20, 26)] and downstream effectors, be they proinflammatory [e.g., IL-8 (20), S100A8 (21)] or anti-inflammatory [e.g., IL-10 (4)]. Besides, Fang et al. (8) have shown that CFTR dysfunction in distal airways leads to altered fluid absorption in mouse and human lung after birth. By using fluid and mRNA from subsequent series of grafts, we will determine whether the inflammatory phenotype of long-term CF distal grafts involves altered volume, composition, or bioactivity of secretions or altered epithelium/leukocyte interactions.
In a previous study, we showed a basal proinflammatory imbalance in CF proximal airway grafts compared with controls, as shown by increased luminal IL-8 levels and abnormal mucosal localization of both human and murine leukocytes (22). Yet, in the absence of infection, no intraluminal inflammation was observed, even when mature CF proximal grafts had a prolonged engraftment time (up to 50 wk). Another puzzling difference lies in the fact that CF and non-CF proximal grafts showed clear intraepithelial localization of leukocytes (22), whereas no intraepithelial leukocytes were observed here in any of the distal grafts studied, even the long-term CF group with intraluminal inflammation. These discrepancies likely reflect three facts. First, the proximal lung epithelium is a normal homing niche for a defined pool of resident leukocytes, whereas the distal lung epithelium may not be. Second, the monostratified distal lung epithelium may allow transmigrating leukocytes to travel swiftly across, but it may otherwise prevent transmigrating cells from staying within the epithelial layer. Third, proximal airways may be endowed with more potent means than distal airways to inhibit leukocyte migration into the lumen when no insult is exerted (even if intraluminal cytokine levels are altered). In further work, we will explore the differences that exist between proximal and distal CF grafts in SCID mice, which may well recapitulate the differential involvement of proximal vs. distal airways in CF patients in vivo (3, 7).
In essence, our study offers the first account of a dysregulated intraluminal recruitment of inflammatory cells in an in vivo model of mature and naïve human CF lung. On the basis of our results and on converging evidence from other groups (21, 26), we believe that, for CF lung pathophysiology to be better understood and treated (15), more effort should be geared at deciphering and counteracting the early inflammatory alterations associated with the disease.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. M. Catala, A.-L. Delezoide, C. Ferec, F. Menez, F. Narcy, J. Martinovic, and J. Tantau for collaboration, as well as Prof. J. Wine, Prof. R. Moss, and Dr. E. Puchelle for helpful suggestions.
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FOOTNOTES |
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This work was funded by grants from CNRS, Institut National de la Santé et de la Recherche Médicale, and Vaincre La Mucoviscidose, the French association against cystic fibrosis.
Address for reprint requests and other correspondence: B. Péault, INSERM U506, Batiment Lavoisier, Groupe Hospitalier Paul Brousse, 12 Av Paul Vaillant-Couturier, 94807 Villejuif Cedex, France (E-mail: U506{at}infobiogen.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 15, 2002;10.1152/ajplung.00419.2001
Received 25 October 2001; accepted in final form 11 March 2002.
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