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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
Uhal BD, Nguyen H. The Witschi Hypothesis revisited after 35 this author at least, to have been forgotten or, worse, ignored. For
years: genetic proof from SP-C BRICHOS domain mutations. Am J reasons unclear but likely not in any way disrespectful to Dr.
Physiol Lung Cell Mol Physiol 305: L906 –L911, 2013. First pub- Haschek, in the years and decades following the initial publica-
lished October 18, 2013; doi:10.1152/ajplung.00246.2013.—Over 35
tion, the ideas first discussed in this seminal paper became known
years ago, Wanda Haschek and Hanspeter Witschi published a theory
for the pathogenesis of lung fibrosis that dared to challenge the and referred to as the “Witschi Hypothesis,” to be discussed in
longstanding view of lung fibrosis as an “inflammatory disease.” On more detail below.
the basis of considerable experimental evidence, they proposed that The writing of this Perspective was stimulated by two recent
lung fibrosis was initiated and propagated by microfoci of epithelial events that I (B. D. Uhal) would like to share. For many years
damage that, if unrepaired, upset the normal epithelial-fibroblast I regularly attended the biennial ICLAF meeting, the last
balance to create profibrotic microenvironments, without any obliga- edition of which was held in Modena, Italy, October 2012
tory contribution of “inflammatory” cells. Unfortunately, this theory under the superb organization and hospitality of Prof. Luca
was largely overlooked for many years. In the meantime, the repeated
Richeldi of the University of Modena and Reggio Emilia. At
failure of attempts to treat idiopathic pulmonary fibrosis with anti-
inflammatory regimens has led some investigators to revive the theory the 2012 edition, the organizers held successful and informa-
referred to, in decades past, as “The Witschi Hypothesis.” This tive breakout sessions at which all attendees split into two
manuscript briefly reviews more recent evidence in support of the groups to debate, among other topics, the “inflammatory” vs.
“Severity of Epithelial Injury” Hypothesis proposed by Haschek and “epithelial” theories described above. Shortly after the debate
Witschi. More important, it offers the updated viewpoint that muta- began, I joined the discussion and asked my breakout group of
tions in the BRICHOS domain of surfactant protein C, which cause approximately 50 persons, “How many people in the room
interstitial lung disease and induce cell death specifically in lung remember the Witschi Hypothesis?” To my astonishment and
epithelial cells, in effect provide genetic proof that the Witschi
dismay, only two hands arose from a room filled with scien-
Hypothesis is indeed the correct theory to explain the pathogenesis of
fibrosis in the lungs. tists, all of whom are currently working on lung fibrogenesis
research. I remember at that moment hoping sincerely that
lung fibrosis; idiopathic pulmonary fibrosis; alveolar epithelial cells; some of those who did not raise their hands were just asleep,
type II pneumocyte; epithelial-mesenchymal cross talk perhaps from excess grappa the previous evening, or perhaps
simply didn’t remember the name associated with the ground-
THIS ARTICLE IS INTENDED TO address one side, which we believe is breaking work to which I had referred. My short recital of the
the correct side, of a debate that continues to be held each year at ideas that Haschek and Witschi so elegantly discovered was
the annual American Thoracic Society (ATS) and European greeted with approving nods from the two souls who raised
Respiratory Society (ERS) meetings, and every other year at the their hands, but blank stares from most others.
International Colloquium on Lung and Airway Fibrosis (ICLAF). That memory planted in my head the seed for this writing,
The debate concerns the pathogenesis of pulmonary fibrosis, but it germinated the following May at the ATS International
which was for many years believed to be an “inflammatory Congress in Philadelphia, where similar debates were held,
disease.” As will be reviewed briefly below, there is much evi- again, with both sides of the debate “agreeing to disagree” as
dence against this traditional view. The correct side of this debate, usual. But the single event that galvanized my resolve to write
which has been slowly gaining acceptance, is the alternate concept was my meeting of a young scientist, who shall go unnamed
that lung fibrogenesis is initiated and propagated by alveolar here, who presented an excellent and well-attended experimen-
epithelial “disrepair,” “activation,” or simply “death,” regardless tal study. Among his findings was a set of data that, in his
of the presence or absence of inflammation. To those readers who words, appeared to “very surprisingly, dissociate the processes
consider the origin of this concept to be relatively recent, it is not: of inflammation and fibrogenesis in the lungs.” My immediate
it was proposed over 35 years ago by Haschek and Witschi (15) response was to ask “Have you ever read the work of Haschek
on the basis of considerable experimental evidence that seems, to and Witschi?,” to which I received a simple “Who?” At that
moment I realized that many investigators in the pulmonary
Address for reprint requests and other correspondence: B. D. Uhal, Dept. of
research community need to be reminded, and young research-
Physiology, Michigan State Univ., 3197 Biomedical and Physical Sciences ers informed, of this seminal work that took place in the late
Bldg., East Lansing, MI 48824 (e-mail: uhal@msu.edu). 1970s and early-mid 1980s.
L906 1040-0605/13 Copyright © 2013 the American Physiological Society http://www.ajplung.org
Perspectives
PROOF THAT EPITHELIAL INJURY CAUSES LUNG FIBROSIS L907
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
A remembrance of the Witschi Hypothesis is extremely of anti-inflammatory clinical trials in IPF. Other authors have
important not only as an academic exercise, but also in light of previously reviewed the evidence against the “inflammation
the repeated failure of anti-inflammatory/immunosuppressive hypothesis” in a clinical perspective and in far more detail than
therapeutic clinical trials to show benefit for the treatment of is possible here (11, 31).
the most insidious and frequent of the interstitial lung diseases, Two counterarguments that proponents of the inflammation
idiopathic pulmonary fibrosis (IPF). The traditional attempts at hypothesis have often raised, at least in the experience of this
treatment of IPF with corticosteroids were ultimately found to author (B. D. Uhal), are 1) that the correct combination of
provide no survival benefit (24) and to be associated with anti-inflammatories for use in IPF has just not yet been found,
significant comorbidity (9). Despite promising preliminary and 2) that IPF patients present late in the course of their
data, the addition of azathioprine to the corticosteroid regimen disease, and therefore it might be possible that anti-inflamma-
did not produce the expected statistically significant improve- tories could provide benefit if administered earlier. Regarding
ment (29). Recently, in a larger meta-analysis, the formerly the latter counterargument, two facts undermine this rationale:
standard “triple therapy” of prednisone, azathioprine, and first, the histopathology of IPF is heterogeneous both spatially
N-acetylcysteine was deemed not only ineffective, but indeed and temporally, often revealing both old and “nascent” lesions,
harmful, on the basis that IPF patients receiving the triple which are believed to reflect “early” pathogenic processes even
therapy experienced more hospitalizations, more adverse con- in patients with more advanced disease (31). If inflammation
sequences, and excess deaths compared with those receiving drove the nascent lesion, anti-inflammatories should still have
the placebo (46). Clearly, the anti-inflammatory/immunosup- provided benefit. Second, although it is very difficult, if not
pressive approach to the treatment of IPF was failing. impossible, to know what exact events initiate the fibrogenic
From our viewpoint, the failure of anti-inflammatory thera- response in patients with IPF, animal models in which exper-
pies in IPF was not entirely surprising; the results of animal imental treatments were administered prophylactically gave
model experiments designed to prove that inflammation, or results that at best conflicted and at worst contradicted a role
specific cells involved in the inflammatory response, played a for inflammation in lung fibrogenesis as discussed above.
causal role in lung fibrosis have not been overly convincing. In Regarding the first counterargument that an optimum anti-
fact, two studies have suggested just the opposite of what the inflammatory cocktail has not yet been constructed, a search of
authors initially predicted and indeed support the alternate the symposia on Clinical Trials in IPF at the annual ATS and
interpretation that inflammation inhibits, not stimulates, sub- ERS meetings over the last 20 years offers evidence that the
sequent fibrogenesis in the lungs. best clinical researchers have devoted considerable effort to
One of the earliest was conducted by Thrall and colleagues testing the most likely candidate anti-inflammatory regimens in
(37), who tested the theory that the influx of neutrophils IPF, albeit unsuccessfully. We suggest that the potential role of
(PMNs) into the lungs shortly after administration of bleomy- pharmaceutical firms in perpetuating these efforts should be
cin to rats played a pivotal role in mediating the subsequent considered carefully and that caution be taken in the planning
deposition of collagens. Using anti-PMN serum developed of future clinical trials of new anti-inflammatory compounds or
in rabbits, they first depleted over 90% of circulating PMNs cocktails.
from rats and then administered bleomycin to induce a fibrotic
response. To the authors’ surprise, the PMN depletion did not Review of the Witschi Hypothesis
reduce, but rather increased, the deposition of lung collagens
significantly, leading them to conclude that the infiltrating Surprisingly to this author, the speculation that lung fibrosis
“PMN, rather than accelerating or promoting the fibrotic re- might not be an inflammatory disease continues to raise eye-
sponse, may be attempting to restrict it” (37). brows, despite the failure of anti-inflammatory/immunosup-
Another is the more recent generation of mice with a more pressive therapeutics in IPF and conflicting experimental data
robust inflammatory response by knockout of the IL-10 gene just mentioned. Yet more than 35 years ago a theory of lung
by Huaux et al. (17). The mice deficient in IL-10, a primarily fibrogenesis that was independent of inflammation was pro-
anti-inflammatory cytokine, had a more robust inflammatory posed and defended on the basis of cleverly designed toxico-
response after administration of silica to the lungs, as demon- logical studies in a variety of animal species as well as lung
strated by increased numbers of lymphocytes, PMNs, lactate explant and cell culture/coculture models (see Fig. 1). The
dehydrogenase (LDH), and total protein in bronchoalveolar earliest of these, published in 1979 by Haschek and Witschi
lavage fluid after the silica challenge, relative to wild-type (15), studied BALB/c mice exposed to precisely titrated doses
mice after challenge. Rather than increasing the deposition of of butylated hydroxytoluene (BHT) followed by carefully
collagens, the more robust inflammatory response imparted by timed and titrated exposures to hyperoxic gas or room air. The
IL-10 knockout significantly decreased lung collagen deposi- antioxidant BHT damaged exclusively the type I alveolar
tion, leading the authors to conclude that “IL-10 is anti- epithelial cell layer, which, without subsequent exposure to
inflammatory but exerts a pro-fibrotic activity” (17). hyperoxia, was replaced by type II pneumocytes proliferating
We suspect that some readers are now frustrated with what and differentiating into type I cells (verified by electron mi-
was just presented, an admittedly incomplete treatment of the croscopy autoradiography) without the induction of fibrosis.
many studies of other inflammatory cell types and processes, If, however, the type II cell division was delayed by addi-
cytokines, and other immune/inflammatory factors that suggest tional exposure to hyperoxic gas, lung collagen deposition
positive and causal roles in lung fibrogenesis. On the other occurred in regions where the epithelial damage was not
hand, we suggest that when considered as a whole, the evi- repaired. Importantly, the hyperoxia itself was not fibrogenic
Injury to Epithelium
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 at the time, this theory predicted a number of suspected epithelial-
before or after the peak of the type II cell proliferative re- fibroblast interactions and regulatory factors that, over the subse-
sponse; the hyperoxia was fibrogenic only if it was applied at quent years, were ultimately discovered to be produced by and
a time and dose that inhibited type II cell proliferation and active in human lung epithelial/fibroblast cross talk. These in-
repair. These observations were the first to lead to the hypoth- clude, but are by no means limited to, hepatocyte growth factor
esis that a lack of alveolar epithelial repair was the key event (HGF) and keratinocyte growth factor (KGF, 27), insulin-like
that led to a fibrotic response. The same conclusion was growth factor I (IGF-1, 34), PGE2 (7), direct epithelial-fibroblast
derived from subsequent studies of blood-free lung explants contacts (5), angiotensin II (45), and hydrogen peroxide (43), to
conducted in the following years by Adamson, Young, Bow- name a few.
den, and others at the University of Manitoba (4). Studying a
wide range of exposure times and concentrations of hyperoxic Abnormal Epithelial-Fibroblast Balance in Lung
gas applied in vivo, these investigators noted that fibrosis in
blood-free lung explants occurred only at locations and expo- The consideration of lung fibrosis as a disease of epithelial-
sure conditions in which alveolar epithelial repair was incom- fibroblast imbalance was revived more recently by Selman et
plete. Detailed electron microscopy and autoradiographic cell al. (31) and Gauldie et al. (11), both of whom did an excellent
kinetic studies supported the hypothesis that the delay of job of articulating the growing evidence against the traditional
epithelial repair, not inflammation, was what led to the activa- view of lung fibrosis as an inflammatory disease. Both groups
tion of nearby fibroblast proliferation and collagen deposition. of authors also reviewed the still-growing experimental evi-
Both the Witschi and Adamson research groups explored this dence describing how the death of epithelial cells, in itself,
hypothesis further over the subsequent decade and longer, with a could create a profibrotic microenvironment without an oblig-
variety of in vivo and ex vivo models and injurious agents atory contribution of inflammatory processes. One of the most
including but not limited to BHT, hyperoxia, paraquat, bleomycin, compelling reviews on this topic, however, was published
silica, and asbestos (1, 3, 14, 16, 20). Those labor-intensive and years earlier by Simon (32), who first discussed the concept of
time-consuming studies, too numerous to review here, consis- “antifibrotic functions” of intact alveolar type I and II cells that
tently supported the same theory that both groups refined in include, among many other roles, 1) robust production of
articles published in later years (2, 49): the hypothesis that the PGE2 that inhibits fibroblast proliferation; 2) constitutive pro-
inability of the epithelium to repair, in itself, upsets the normal duction of plasminogen activators that help to degrade micro-
epithelial-fibroblast balance and thereby creates a profibrotic mi- foci of fibrin, along which fibroblasts can migrate into the
croenvironment sufficient to promote lung collagen deposition, alveolar air spaces; 3) production and deposition of basement
regardless of the presence or absence of inflammation. A new idea membrane components (laminins, fibronectins) that promote
Table 1. Fibrogenic SP-C BRICHOS domain mutations and alveolar epithelial cell death
Mutation in sequence order G100S P115L Exon 4 A116D Q145H T187N L188P L188Q C189Y L194P
Causes ILD yes yes yes yes yes yes yes yes yes yes
Cause AEC Death yes ? yes yes ? ? ? yes ? ?
UPR markers increased ? increased ? ? ? ? increased ? ?
ER stress Bip, HSPs increased reduced (38) increased reduced (38) ? ? ? increased (19) ? ?
increased (50) reduced (38)
References 26, 42 25, 38, 48 21, 23 38, 50 12 48 12 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.