Am J Physiol Lung Cell Mol Physiol 305: L906–L911, 2013.
First published October 18, 2013; doi:10.1152/ajplung.00246.2013.
Perspectives
Translational Research in Acute Lung Injury
and Pulmonary Fibrosis
CALL FOR PAPERS
The Witschi Hypothesis revisited after 35 years: genetic proof from SP-C
BRICHOS domain mutations
Bruce D. Uhal1 and Hang Nguyen2
1
Department of Physiology and 2Department of Biochemistry, Michigan State University, East Lansing, Michigan
Submitted 3 September 2013; accepted in final form 15 October 2013
lung fibrosis; idiopathic pulmonary fibrosis; alveolar epithelial cells;
type II pneumocyte; epithelial-mesenchymal cross talk
THIS ARTICLE IS INTENDED TO address one side, which we believe is
the correct side, of a debate that continues to be held each year at
the annual American Thoracic Society (ATS) and European
Respiratory Society (ERS) meetings, and every other year at the
International Colloquium on Lung and Airway Fibrosis (ICLAF).
The debate concerns the pathogenesis of pulmonary fibrosis,
which was for many years believed to be an “inflammatory
disease.” As will be reviewed briefly below, there is much evidence against this traditional view. The correct side of this debate,
which has been slowly gaining acceptance, is the alternate concept
that lung fibrogenesis is initiated and propagated by alveolar
epithelial “disrepair,” “activation,” or simply “death,” regardless
of the presence or absence of inflammation. To those readers who
consider the origin of this concept to be relatively recent, it is not:
it was proposed over 35 years ago by Haschek and Witschi (15)
on the basis of considerable experimental evidence that seems, to
Address for reprint requests and other correspondence: B. D. Uhal, Dept. of
Physiology, Michigan State Univ., 3197 Biomedical and Physical Sciences
Bldg., East Lansing, MI 48824 (e-mail: [email protected]).
L906
this author at least, to have been forgotten or, worse, ignored. For
reasons unclear but likely not in any way disrespectful to Dr.
Haschek, in the years and decades following the initial publication, the ideas first discussed in this seminal paper became known
and referred to as the “Witschi Hypothesis,” to be discussed in
more detail below.
The writing of this Perspective was stimulated by two recent
events that I (B. D. Uhal) would like to share. For many years
I regularly attended the biennial ICLAF meeting, the last
edition of which was held in Modena, Italy, October 2012
under the superb organization and hospitality of Prof. Luca
Richeldi of the University of Modena and Reggio Emilia. At
the 2012 edition, the organizers held successful and informative breakout sessions at which all attendees split into two
groups to debate, among other topics, the “inflammatory” vs.
“epithelial” theories described above. Shortly after the debate
began, I joined the discussion and asked my breakout group of
approximately 50 persons, “How many people in the room
remember the Witschi Hypothesis?” To my astonishment and
dismay, only two hands arose from a room filled with scientists, all of whom are currently working on lung fibrogenesis
research. I remember at that moment hoping sincerely that
some of those who did not raise their hands were just asleep,
perhaps from excess grappa the previous evening, or perhaps
simply didn’t remember the name associated with the groundbreaking work to which I had referred. My short recital of the
ideas that Haschek and Witschi so elegantly discovered was
greeted with approving nods from the two souls who raised
their hands, but blank stares from most others.
That memory planted in my head the seed for this writing,
but it germinated the following May at the ATS International
Congress in Philadelphia, where similar debates were held,
again, with both sides of the debate “agreeing to disagree” as
usual. But the single event that galvanized my resolve to write
was my meeting of a young scientist, who shall go unnamed
here, who presented an excellent and well-attended experimental study. Among his findings was a set of data that, in his
words, appeared to “very surprisingly, dissociate the processes
of inflammation and fibrogenesis in the lungs.” My immediate
response was to ask “Have you ever read the work of Haschek
and Witschi?,” to which I received a simple “Who?” At that
moment I realized that many investigators in the pulmonary
research community need to be reminded, and young researchers informed, of this seminal work that took place in the late
1970s and early-mid 1980s.
1040-0605/13 Copyright © 2013 the American Physiological Society
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Uhal BD, Nguyen H. The Witschi Hypothesis revisited after 35
years: genetic proof from SP-C BRICHOS domain mutations. Am J
Physiol Lung Cell Mol Physiol 305: L906 –L911, 2013. First published October 18, 2013; doi:10.1152/ajplung.00246.2013.—Over 35
years ago, Wanda Haschek and Hanspeter Witschi published a theory
for the pathogenesis of lung fibrosis that dared to challenge the
longstanding view of lung fibrosis as an “inflammatory disease.” On
the basis of considerable experimental evidence, they proposed that
lung fibrosis was initiated and propagated by microfoci of epithelial
damage that, if unrepaired, upset the normal epithelial-fibroblast
balance to create profibrotic microenvironments, without any obligatory contribution of “inflammatory” cells. Unfortunately, this theory
was largely overlooked for many years. In the meantime, the repeated
failure of attempts to treat idiopathic pulmonary fibrosis with antiinflammatory regimens has led some investigators to revive the theory
referred to, in decades past, as “The Witschi Hypothesis.” This
manuscript briefly reviews more recent evidence in support of the
“Severity of Epithelial Injury” Hypothesis proposed by Haschek and
Witschi. More important, it offers the updated viewpoint that mutations in the BRICHOS domain of surfactant protein C, which cause
interstitial lung disease and induce cell death specifically in lung
epithelial cells, in effect provide genetic proof that the Witschi
Hypothesis is indeed the correct theory to explain the pathogenesis of
fibrosis in the lungs.
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PROOF THAT EPITHELIAL INJURY CAUSES LUNG FIBROSIS
The Failure of Anti-Inflammatory Therapies
dence supporting the view of lung fibrosis as an inflammatory
disease is at best conflicting, particularly in light of the failure
of anti-inflammatory clinical trials in IPF. Other authors have
previously reviewed the evidence against the “inflammation
hypothesis” in a clinical perspective and in far more detail than
is possible here (11, 31).
Two counterarguments that proponents of the inflammation
hypothesis have often raised, at least in the experience of this
author (B. D. Uhal), are 1) that the correct combination of
anti-inflammatories for use in IPF has just not yet been found,
and 2) that IPF patients present late in the course of their
disease, and therefore it might be possible that anti-inflammatories could provide benefit if administered earlier. Regarding
the latter counterargument, two facts undermine this rationale:
first, the histopathology of IPF is heterogeneous both spatially
and temporally, often revealing both old and “nascent” lesions,
which are believed to reflect “early” pathogenic processes even
in patients with more advanced disease (31). If inflammation
drove the nascent lesion, anti-inflammatories should still have
provided benefit. Second, although it is very difficult, if not
impossible, to know what exact events initiate the fibrogenic
response in patients with IPF, animal models in which experimental treatments were administered prophylactically gave
results that at best conflicted and at worst contradicted a role
for inflammation in lung fibrogenesis as discussed above.
Regarding the first counterargument that an optimum antiinflammatory cocktail has not yet been constructed, a search of
the symposia on Clinical Trials in IPF at the annual ATS and
ERS meetings over the last 20 years offers evidence that the
best clinical researchers have devoted considerable effort to
testing the most likely candidate anti-inflammatory regimens in
IPF, albeit unsuccessfully. We suggest that the potential role of
pharmaceutical firms in perpetuating these efforts should be
considered carefully and that caution be taken in the planning
of future clinical trials of new anti-inflammatory compounds or
cocktails.
Review of the Witschi Hypothesis
Surprisingly to this author, the speculation that lung fibrosis
might not be an inflammatory disease continues to raise eyebrows, despite the failure of anti-inflammatory/immunosuppressive therapeutics in IPF and conflicting experimental data
just mentioned. Yet more than 35 years ago a theory of lung
fibrogenesis that was independent of inflammation was proposed and defended on the basis of cleverly designed toxicological studies in a variety of animal species as well as lung
explant and cell culture/coculture models (see Fig. 1). The
earliest of these, published in 1979 by Haschek and Witschi
(15), studied BALB/c mice exposed to precisely titrated doses
of butylated hydroxytoluene (BHT) followed by carefully
timed and titrated exposures to hyperoxic gas or room air. The
antioxidant BHT damaged exclusively the type I alveolar
epithelial cell layer, which, without subsequent exposure to
hyperoxia, was replaced by type II pneumocytes proliferating
and differentiating into type I cells (verified by electron microscopy autoradiography) without the induction of fibrosis.
If, however, the type II cell division was delayed by additional exposure to hyperoxic gas, lung collagen deposition
occurred in regions where the epithelial damage was not
repaired. Importantly, the hyperoxia itself was not fibrogenic
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A remembrance of the Witschi Hypothesis is extremely
important not only as an academic exercise, but also in light of
the repeated failure of anti-inflammatory/immunosuppressive
therapeutic clinical trials to show benefit for the treatment of
the most insidious and frequent of the interstitial lung diseases,
idiopathic pulmonary fibrosis (IPF). The traditional attempts at
treatment of IPF with corticosteroids were ultimately found to
provide no survival benefit (24) and to be associated with
significant comorbidity (9). Despite promising preliminary
data, the addition of azathioprine to the corticosteroid regimen
did not produce the expected statistically significant improvement (29). Recently, in a larger meta-analysis, the formerly
standard “triple therapy” of prednisone, azathioprine, and
N-acetylcysteine was deemed not only ineffective, but indeed
harmful, on the basis that IPF patients receiving the triple
therapy experienced more hospitalizations, more adverse consequences, and excess deaths compared with those receiving
the placebo (46). Clearly, the anti-inflammatory/immunosuppressive approach to the treatment of IPF was failing.
From our viewpoint, the failure of anti-inflammatory therapies in IPF was not entirely surprising; the results of animal
model experiments designed to prove that inflammation, or
specific cells involved in the inflammatory response, played a
causal role in lung fibrosis have not been overly convincing. In
fact, two studies have suggested just the opposite of what the
authors initially predicted and indeed support the alternate
interpretation that inflammation inhibits, not stimulates, subsequent fibrogenesis in the lungs.
One of the earliest was conducted by Thrall and colleagues
(37), who tested the theory that the influx of neutrophils
(PMNs) into the lungs shortly after administration of bleomycin to rats played a pivotal role in mediating the subsequent
deposition of collagens. Using anti-PMN serum developed
in rabbits, they first depleted over 90% of circulating PMNs
from rats and then administered bleomycin to induce a fibrotic
response. To the authors’ surprise, the PMN depletion did not
reduce, but rather increased, the deposition of lung collagens
significantly, leading them to conclude that the infiltrating
“PMN, rather than accelerating or promoting the fibrotic response, may be attempting to restrict it” (37).
Another is the more recent generation of mice with a more
robust inflammatory response by knockout of the IL-10 gene
by Huaux et al. (17). The mice deficient in IL-10, a primarily
anti-inflammatory cytokine, had a more robust inflammatory
response after administration of silica to the lungs, as demonstrated by increased numbers of lymphocytes, PMNs, lactate
dehydrogenase (LDH), and total protein in bronchoalveolar
lavage fluid after the silica challenge, relative to wild-type
mice after challenge. Rather than increasing the deposition of
collagens, the more robust inflammatory response imparted by
IL-10 knockout significantly decreased lung collagen deposition, leading the authors to conclude that “IL-10 is antiinflammatory but exerts a pro-fibrotic activity” (17).
We suspect that some readers are now frustrated with what
was just presented, an admittedly incomplete treatment of the
many studies of other inflammatory cell types and processes,
cytokines, and other immune/inflammatory factors that suggest
positive and causal roles in lung fibrogenesis. On the other
hand, we suggest that when considered as a whole, the evi-
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PROOF THAT EPITHELIAL INJURY CAUSES LUNG FIBROSIS
Injury to Epithelium
Normal Epithelial Repair
Basement
membrane
ATII
ATI
Recuperation
Fibrosis
Fig. 1. Summary of the “Severity of Epithelial Injury” or “Witschi” Hypothesis proposed in 1979 by Wanda Haschek and Hanspeter Witschi (15). Microfoci
of alveolar epithelial injury, which often impact the more sensitive alveolar epithelial type I cells (ATI), are normally repaired by type II pneumocyte (ATII)
proliferation without fibrosis (blue). If alveolar epithelial repair by ATII cells is delayed (red), underlying fibroblasts are released from normal epithelial
inhibitions and fibrosis progresses. This theory was supported by robust experimental evidence (1, 2, 3, 14, 15, 16, 20, 49). More recently discovered fibrogenic
surfactant protein C BRICHOS domain mutations that delay epithelial repair by inducing ATII cell apoptosis now offer genetic confirmation of this hypothesis.
See text for details.
when applied alone, nor if applied together with BHT at times
before or after the peak of the type II cell proliferative response; the hyperoxia was fibrogenic only if it was applied at
a time and dose that inhibited type II cell proliferation and
repair. These observations were the first to lead to the hypothesis that a lack of alveolar epithelial repair was the key event
that led to a fibrotic response. The same conclusion was
derived from subsequent studies of blood-free lung explants
conducted in the following years by Adamson, Young, Bowden, and others at the University of Manitoba (4). Studying a
wide range of exposure times and concentrations of hyperoxic
gas applied in vivo, these investigators noted that fibrosis in
blood-free lung explants occurred only at locations and exposure conditions in which alveolar epithelial repair was incomplete. Detailed electron microscopy and autoradiographic cell
kinetic studies supported the hypothesis that the delay of
epithelial repair, not inflammation, was what led to the activation of nearby fibroblast proliferation and collagen deposition.
Both the Witschi and Adamson research groups explored this
hypothesis further over the subsequent decade and longer, with a
variety of in vivo and ex vivo models and injurious agents
including but not limited to BHT, hyperoxia, paraquat, bleomycin,
silica, and asbestos (1, 3, 14, 16, 20). Those labor-intensive and
time-consuming studies, too numerous to review here, consistently supported the same theory that both groups refined in
articles published in later years (2, 49): the hypothesis that the
inability of the epithelium to repair, in itself, upsets the normal
epithelial-fibroblast balance and thereby creates a profibrotic microenvironment sufficient to promote lung collagen deposition,
regardless of the presence or absence of inflammation. A new idea
at the time, this theory predicted a number of suspected epithelialfibroblast interactions and regulatory factors that, over the subsequent years, were ultimately discovered to be produced by and
active in human lung epithelial/fibroblast cross talk. These include, but are by no means limited to, hepatocyte growth factor
(HGF) and keratinocyte growth factor (KGF, 27), insulin-like
growth factor I (IGF-1, 34), PGE2 (7), direct epithelial-fibroblast
contacts (5), angiotensin II (45), and hydrogen peroxide (43), to
name a few.
Abnormal Epithelial-Fibroblast Balance in Lung
The consideration of lung fibrosis as a disease of epithelialfibroblast imbalance was revived more recently by Selman et
al. (31) and Gauldie et al. (11), both of whom did an excellent
job of articulating the growing evidence against the traditional
view of lung fibrosis as an inflammatory disease. Both groups
of authors also reviewed the still-growing experimental evidence describing how the death of epithelial cells, in itself,
could create a profibrotic microenvironment without an obligatory contribution of inflammatory processes. One of the most
compelling reviews on this topic, however, was published
years earlier by Simon (32), who first discussed the concept of
“antifibrotic functions” of intact alveolar type I and II cells that
include, among many other roles, 1) robust production of
PGE2 that inhibits fibroblast proliferation; 2) constitutive production of plasminogen activators that help to degrade microfoci of fibrin, along which fibroblasts can migrate into the
alveolar air spaces; 3) production and deposition of basement
membrane components (laminins, fibronectins) that promote
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Basement
membrane
Delayed Epithelial Repair
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PROOF THAT EPITHELIAL INJURY CAUSES LUNG FIBROSIS
death and, as predicted by Witschi, increased lung collagen
deposition in the mice expressing the epithelial DT receptor in
the lungs (but not in wild-type mice). Despite using completely
different methods, all these studies strongly support the conclusions that 1) inducing lung epithelial cell death causes lung
collagen deposition, and 2) inhibiting lung epithelial cell death
blocks the lung collagen deposition that otherwise would
follow an injury to the epithelium. All these outcomes were
predicted by the Witschi Hypothesis.
Evidence that AEC Death Is Sufficient to Cause Lung
Fibrogenesis
SP-C BRICHOS Mutations Induce Epithelial Cell Death and
Lung Fibrogenesis
Currently there exists a surge of interest by the lung fibrosis
research community in the roles of autophagy in lung fibrogenesis. More than a decade ago, a similar interest centered on
the topic of apoptosis, at that time a “new” idea as it related to
lung cells. The observation of apoptosis markers in the alveolar
epithelium of lung biopsies obtained from patients with IPF
(28, 41) led several research groups to test the ideas first
offered by Haschek and Witschi, by utilizing the potential of
pharmacological inhibitors of apoptosis. The caspase inhibitor
ZVADfmk, administered in vivo to mice or rats, potently
inhibited apoptosis of alveolar epithelial cells after bleomycin
administration as expected, but more importantly it also
blocked the subsequent accumulation of lung collagens (18,
44) as predicted by the Witschi Hypothesis. Moreover, the
discovery that alveolar epithelial cells could express the receptor Fas (Apo1, CD95) after lung injury (8) led to the design of
experiments to test whether induction of apoptosis, directed
toward the epithelium by intratracheal administration of Fas
activators, could produce a fibrogenic response. As expected,
tracheal administrations of Fas-activating antibodies induced
apoptosis mainly in the lung epithelium that was followed by
collagen accumulation several weeks later (13). The effect was
also dose dependent, since greater induction of epithelial apoptosis with higher doses of Fas activator led to greater collagen
accumulation.
One criticism of these studies was the potential nonspecificity of caspase inhibitors or Fas activators. However, two
different genetic approaches produced the same result: germline knockout of the apoptosis signaling molecule Bid in mice
prevented TGF-␤1-induced epithelial apoptosis and the deposition of lung collagens that would otherwise follow intratracheal bleomycin administration (6). Another completely different approach, but that again yielded the same outcome, was
taken by Sisson et al. (33), who used the surfactant protein C
(SP-C) promoter to express the human diphtheria toxin (DT)
receptor specifically in the alveolar epithelium of mice. Subsequent administration of DT induced alveolar epithelial cell
At least 10 pathogenic mutations in the BRICHOS domain of
the human SP-C have now been discovered through genetic
analyses of DNA samples from infants, children, and/or adults
with diffuse interstitial lung diseases (see Table 1). As described
in detail by their discoverers (19, 22, 26), the BRICHOS domain
is involved in processing of the immature SP-C and, in particular,
its proper folding during the intracellular processing steps that
eventually, under normal conditions, led to the correct packaging
of the mature SP-C together with other surfactant components into
the lamellar bodies for secretion. BRICHOS domain mutations
can disrupt protein folding.
Important for the discussion here is the cell-specific expression of SP-C by the type II pneumocyte, the only cell type in
the body that expresses this particular SP. Indeed, the human
SP-C promoter has long been used as the “promoter of choice”
for the design of transgenes to be expressed specifically in lung
alveolar epithelial cells (AECs, 47). More important, however,
are the findings (see Table 1) that for each SP-C mutation that
has been evaluated in the following respect, all of them have
one mechanism in common: the induction of cell death in the
AEC secondary to SP-C misfolding caused by the mutation. In
the three SP-C mutants best studied to date (G100S, L188Q,
and exon4), the type of cell death appears to be apoptosis
signaled through the unfolded protein response (UPR) by
specific pathways currently being identified. Although most,
but not all, of the fibrogenic SP-C mutations increase endoplasmic reticulum (ER) stress markers such as Bip or HSP-70
or -90 when transfected into A549 or MLE cells, it is likely that
apoptosis is signaled by the UPR rather than ER stress per se
(21). Some of the mutations listed in Table 1 have been
reported to have no effect on cell viability measured by MTT
assay (38), but apoptosis was not specifically evaluated in
those studies. This is particularly important in studies of
epithelial cells, in light of the peculiar ability of epithelial
barrier cells to undergo high rates of apoptosis without compromising barrier integrity, i.e., without the collapse of plasma
membrane integrity required for assays such as LDH release or
Table 1. Fibrogenic SP-C BRICHOS domain mutations and alveolar epithelial cell death
Mutation in sequence order
G100S
Causes ILD
Cause AEC Death
UPR markers
ER stress Bip, HSPs
yes
yes
increased
increased
References
26, 42
P115L
Exon 4
?
?
reduced (38)
yes
yes
increased
increased
25, 38, 48
21, 23
yes
A116D
yes
yes
?
reduced (38)
increased (50)
38, 50
Q145H
T187N
L188P
L188Q
C189Y
L194P
yes
?
?
?
yes
?
?
?
yes
?
?
?
yes
?
?
?
yes
?
?
?
12
48
12
yes
yes
increased
increased (19)
reduced (38)
19, 22, 38
12, 36
12
SP-C, surfactant protein C; ILD, interstitial lung disease; AEC, alveolar epithelial cell; UPR, unfolded protein response; ER, endoplasmic reticulum; HSPs,
heat shock proteins.
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epithelial rather than mesenchymal differentiation signaling;
and, more recently discovered, 4) generation of matrix metalloproteinases of the subtypes that degrade interstitial collagens
(10). Owing to these and other antifibrotic activities of intact
alveolar epithelial cells, death or incomplete repair of a normal
epithelial layer could be sufficient to initiate and support
fibrogenesis. We refer the reader to those and other excellent
reviews for a much more complete treatment of this important
topic.
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PROOF THAT EPITHELIAL INJURY CAUSES LUNG FIBROSIS
dye exclusion (40). Therefore, determinations of whether the
other fibrogenic SP-C mutants listed in Table 1 also induce
apoptosis, or any other mode of AEC death, will be a most
important topic in light of the seminal theory of Dr. Haschek
and Dr. Witschi. We suggest that these determinations be
performed carefully, since LDH release (50) or MTT assay (38)
are not capable of detecting apoptosis, and even a seemingly
minor stimulation of apoptosis that is nonetheless sufficient to
reduce the capacity of the epithelium to repair would compromise
reepithelialization over time (39).
Summary and Perspective
ACKNOWLEDGMENTS
Portions of the work completed by H. Nguyen were performed in partial
fulfillment of the requirements for the PhD degree from Michigan State
University.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
award HL-45136 to B. D. Uhal.
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
B.D.U. conception and design of research; B.D.U. drafted manuscript;
B.D.U. and H.N. edited and revised manuscript; B.D.U. and H.N. approved
final version of manuscript; H.N. analyzed data.
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The above discussion attempts to articulate our rationale
for concluding that epithelial cell death, not inflammation,
“causes” fibrosis in the lungs by upsetting the normally antifibrotic epithelial-fibroblast cross talk. When comparing the
two sides of this debate, every published attempt to initiate
lung fibrosis by inducing cell death, as specifically as possible
in the epithelium, produced a fibrotic response (13, 33). Moreover, every published attempt to inhibit alveolar epithelial cell
death through caspase inhibition (18, 44), genetic deletion of
apoptosis signaling molecules (6), or counteracting cell death
through administrations of epithelial-specific mitogens (30)
resulted in inhibition of lung fibrogenesis. Can a similar argument be made for attempts to test the role of inflammation in
the causation of lung fibrogenesis? The answer is clearly no:
efforts to deplete individual inflammatory cells (37) or to create
a more robust inflammatory response (17) gave results opposite
to those expected and led the authors to conclude that the
inflammatory cells chosen for study appeared to inhibit, not
promote, fibrogenesis as initially hypothesized.
We now know that the BRICHOS domain mutations of
SP-C have several features in common that strongly support
the “Severity of Epithelial Injury” or “Witschi” hypothesis:
1) SP-C is expressed in only one cell type, the alveolar
epithelial type II cell; and 2) in every case in which apoptosis
has been evaluated, fibrogenic SP-C BRICHOS mutations
induce cell death in the alveolar epithelial cell. On this basis
alone, these mutations should be considered genetic proof that
the hypothesis proposed in 1979 by Haschek and Witschi was
correct. Many years ago, a reviewer of one my past grants
suggested that to prove this hypothesis I needed to induce cell
death specifically in the alveolar epithelium and demonstrate a
subsequent fibrotic response. Is this not exactly what nature has
done in the fibrogenic SP-C BRICHOS domain mutants that
induce apoptosis specifically in type II pneumocytes? To supporters of the inflammation hypothesis that still prefer to
disagree, responses to this Perspective are invited. However, it
is asked that rebuttals written to support the inflammation
hypothesis be based mainly on published data, as was done
here, rather than speculation or wishful thinking.
DISCLOSURES
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