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Regulation of Non-Infectious Lung Injury Inflammation and Repair by the Extracellular Matrix Glycosaminoglycan Hyaluronan.

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THE ANATOMICAL RECORD 293:982–985 (2010)
Regulation of Non-Infectious Lung
Injury, Inflammation, and Repair by the
Extracellular Matrix Glycosaminoglycan
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Duke
University School of Medicine, Durham, North Carolina
An important hallmark of tissue remodeling is the dynamic turnover
of extracellular matrix (ECM). ECM performs a variety of functions in tissue repair including scaffold formation, modulation of fluid dynamics, and
regulating cell behavior. During non-infectious tissue injury ECM degradation products are generated that acquire signaling functions not attributable to the native precursor molecules. Hyaluronan (HA) is a nonsulfated glycosaminoglycan which is produced in great abundance following tissue injury. It exists both in a soluble form and as side chains on
proteoglycans. HA has critical roles in development as well as a variety of
biological processes including wound healing, tumor growth and metastasis, and inflammation. HA fragments share structural similarities with
pathogens and following tissue injury can be recognized by innate
immune receptors. Elucidating the protean roles of HA in tissue injury,
inflammation, and repair will generate new insights into mechanisms of
diseases characterized by chronic inflammation and tissue remodeling.
C 2010 Wiley-Liss, Inc.
Anat Rec, 293:982–985, 2010. V
Key words: extracellular matrix; glycosaminoglycan; lung injury
Tissue injury and remodeling occurs in a variety of
settings that can involve infection, inflammation, and
autoimmunity. The mechanisms that regulate tissue
injury and repair following infection have become well
understood, but the critical elements involved in determining the host responses to non-infectious or sterile
inflammation remain unclear. Our laboratory has been
interested in elucidating the mechanisms that contribute
to lung injury, inflammation, and repair following noninfectious fibrotic lung injury. Tissue fibrosis is a leading
cause of morbidity and mortality, and fibrotic lung diseases represent a major area of unmet medical need.
Fibrosing lung diseases that occur in the absence of recognized inciting agents represent areas of investigation
in need of new insights into both pathogenesis and
determinants of progression. The most serious disorder
of lung fibrosis is idiopathic pulmonary fibrosis (IPF)
(Noble and Homer, 2004; Noble, 2006). IPF is a disorder
characterized by unremitting deposition of components
of the extracellular matrix (ECM) in the interstitium of
the lung leading to destruction of gas-exchanging
regions of the lung and ultimate suffocation. The pattern
of deposition of ECM is unique in IPF and a number of
components of the ECM accumulate including collagens,
proteoglycans, fibronections, and the non-sulfated glycosaminoglycan hyaluronan (HA). Unremitting fibrosis
involves the interactions between matrix components
with mesenchymal cells leading to proliferation, tissue
destruction, and further production of matrix.
One of the hallmarks of non-infectious lung injury is
production of components of the ECM, and in recent
years it has become evident that in addition to provide a
substrate for cell migration, matrix can directly influence cell effector functions (Noble, 2002; Jiang et al.,
*Correspondence to: Paul W. Noble, Division of Pulmonary,
Allergy, and Critical Care Medicine, Department of Medicine,
Duke University School of Medicine, 106 Research Drive, Durham, NC 27710. Fax: 919-684-5389.
Received 8 August 2009; Accepted 30 November 2009
DOI 10.1002/ar.21102
Published online 23 February 2010 in Wiley InterScience (www.
Fig. 1. Following bleomycin injury there is a massive accumulation of hyaluronan in both the alveolar
spaces and interstitium of the lung. Lung tissue stained for hyaluronan with biotinylated hyaluronan binding protein at day 14 after bleomycin injury. Wild type (left panel) and CD44-deficient (right panel) mice.
Adapted from Teder et al., 2002.
2007). Our laboratory has focused on the role of HA in
the pathobiology of non-infectious lung injury for a number of reasons. First and foremost, HA is produced in
great abundance following tissue injury. The classic
model for investigating non-infectious fibrotic lung
injury for decades has been the instillation of bleomycin
into the lung (Nettelbladt et al., 1989; Bray et al., 1991;
Teder et al., 2002; Jiang et al., 2005). Bleomycin causes
direct injury to the lung epithelium and orchestrates a
sequence of events characterized by the influx of inflammatory cells, the resolution of inflammation and the development of lung fibrosis that is limited. While the
model does not depict essential features of IPF, it is useful in testing paradigms for resolving and perpetuating
fibrosis. Following bleomycin injury there is a massive
accumulation of HA in both the alveolar spaces and
interstitium of the lung (Teder et al., 2002) (Fig. 1). Our
laboratory has been interested in understanding the significance of this accumulation of matrix. The vast majority of HA is cleared from the lung within 14 days after
injury and that is when the preponderance of collagen
deposition ensues (Teder et al., 2002). Interestingly,
fibroblasts are the main source of both HA and collagen,
and under physiologic conditions HA production ceases
as collagen production increases. HA is non-sulfated
polymer made up of repeating units of D-glucuronic acid
and N-acetyl-glucosamine (Jiang et al., 2007). Under
physiologic conditions in the unchallenged lung, HA
exists as a very large polymer in excess of one million
Daltons. Following tissue injury, there is an accumulation of HA degradation products that subsequently
cleared from the normal lung within 14 days of injury.
Interestingly, the accumulation of HA fragments coincides with the peak inflammatory response. We have
been interested in the biological significance of HA fragment accumulation, particularly since HA has been
shown to accumulate in fibrosing lung diseases. To begin
to understand the potential significance of HA fragment
accumulation, we began to explore the effects of HA
fragments on macrophage functions in vitro (Jiang
et al., 2005). HA has several cell surface receptors
including CD44 and RHAMM, and we have been interested in the role of CD44 in regulating HA interactions
with cells involved in lung disease pathogenesis. CD44
is highly expressed on lung macrophages and ligation of
CD44 has been shown to result in the release of inflammatory mediators. Moreover, Savani and colleagues
showed that the interaction of RHAMM with HA is important in recruitment of macrophages to the lung after
bleomycin induced lung injury (Zaman et al., 2005). We
found that HA fragments produced very different effects
on macrophages than high molecular weight HA (Noble
et al., 1996; Horton et al., 1998). HA fragments induced
a variety of inflammatory mediators that have recognized functions in tissue injury, inflammation, and
repair. In particular, we found that HA fragments
induced the expression of a number of chemokines in
macrophages (Noble et al., 1996; Horton et al., 1998;
Jiang et al., 2005). Chemokines are critical mediators of
inflammatory cell recruitment to sites of tissue injury. In
addition, we found that HA fragments induced the activation of a critical regulator of innate immune
responses, the transcriptional regulator NF-jB (Noble
et al., 1996). This was the first demonstration that a matrix component, modified by the inflammatory milieu
could active a regulatory system that senses host invasion by infectious agents. With these data we began to
explore the concept that HA fragments serve the purpose of signaling that the host has been intruded upon
and may function in analogous role in non-infectious
injury that bacteria do under infectious conditions.
Having discovered that HA fragments could stimulate
inflammatory macrophages to produce mediators of host
repair, we sought to better define the role of CD44 in
regulating HA functions. We had generated data that
CD44 could mediate some aspects of HA fragment signaling such as TNFa and IGF-1 production, but not
others such as MMP-12 (Noble et al., 1993). To explore
the role of CD44 both in vitro and in vivo, we took
advantage of CD44 null mice. These mice develop without obvious impairment and breed normally (Schmits
et al., 1997). We found that macrophages from CD44
null mice still responded to HA fragments suggesting an
alternative cell surface recognition system was involved.
However, when we challenged the CD44 null mice with
bleomycin we found that they had increased susceptibility to non-infectious lung injury (Teder et al., 2002). We
explored this phenotype in great detail and determined
that there was a marked impairment in the ability
of the lung to resolve the inflammatory response.
Fig. 2. Hyaluronan fragments stimulate chemokine expression
through both TLR4 and TLR2. A: Cxcl2 mRNA expression by elicited
peritoneal macrophages from wild type (wt), TLR2 / , TLR4 / , or
TLR2 / 4 / mice treated with HA fragments or LPS in the presence
or absence (underlined) of polymyxin B, detected by Northern analy-
Furthermore, HA fragments were not cleared from the
lung after injury. This impaired clearance resulted in
unremitting inflammation that compromised the host.
Since CD44 is expressed on all cells, we generated bone
marrow chimeras to determine if hematopoietic CD44
was responsible for the inflammatory phenotype. Mice
that expressed CD44 in bone marrow derived cells but
not structural cells were able to clear HA fragments and
resolve the inflammatory response (Teder et al., 2002). It
was evident from these studies that HA and CD44 were
of fundamental importance in mediating the host
response to non-infectious injury. It was also evident
that there must be another recognition system on macrophages that recognized HA fragments.
The clues to the recognition system were in the structure of the HA polymer. HA is a repeating pattern of disaccharides and innate immune cells recognize pathogens
through pattern recognition receptors. The cell surface of
gram positive organisms contain HA and the side chains
of gram-negative organisms also have structural similarities, so we wondered if HA fragments could function like
an infectious agent when generated in the context of the
inflammatory response. The best studied group of pattern
recognition receptors are the Toll-like receptors (TLRs)
(Medzhitov and Janeway, 2000; Akira and Takeda, 2004).
We had been using macrophages from C3H/HeJ mice, for
our in vitro studies, to avoid the effects of contaminating
endotoxins. C3H/HeJ mice are defective in TLR4 signaling
(Poltorak et al., 1998), and so we were skeptical that TLR4
would be the key TLR involved in HA recognition. The
first step in determining if TLRs might be involved was to
utilize MyD88 null macrophages. MyD88 is an important
adaptor for most but not all TLR agonist signaling (Medzhitov and Janeway, 2000; Akira and Takeda, 2004). We
were excited to find out that MyD88 null macrophages did
not respond effectively to HA fragments (Jiang et al.,
2005). This directly indicted the TLR system in HA recognition. We then tested macrophages from TLR1–5 and
TLR9 null mice and found that TLR4 and TLR2 null macrophages had reduced response to HA fragments, and all
other TLRs tested still had similar response to HA fragments as wild type. This was discouraging but we
returned to our concept that perhaps both gram-positive
(TLR2) and gram-negative (TLR4) recognition systems
were necessary. We generated TLR2/TLR4 double knockout mice and stimulated macrophages with HA fragments.
Macrophages from the TLR2/TLR4 double knockout mice
failed to respond to HA fragments (Jiang et al., 2005) (Fig.
sis. B: TNFa protein expression by elicited peritoneal macrophages
from wt or TLR2 / 4 / treated with the indicated concentration of
HA in the presence of polymyxin B for 24 hr. Adapted from Jiang
et al., 2005.
Fig. 3. TLR2/TLR4 null mice had increased susceptibility to non-infectious lung injury. Wild type and TLR2 / 4 / mice were subjected
to intratracheal bleomycin treatment. Percentages of surviving animals
were plotted over a 21-day period. Adapted from Jiang et al., 2005.
2). These data strongly implicated the innate immune system in the recognition of HA fragments. HA-TLR interactions have been demonstrated in dendritic cells (Termeer
et al., 2002), macrophages (Taylor et al., 2007), and microvascular endothelial cells (Taylor et al., 2004). Recently, a
study by Gallo and associates showed that HA fragments
interact with a receptor complex including TLR4, CD44,
and MD-2 in non-infectious injury (Taylor et al., 2007).
This is analogous to LPS-TLR-CD14-MD2 interactions in
infectious injury (Medzhitov and Janeway, 2000).
Having determined that HA fragments were a component of the innate immune response under conditions of
sterile inflammation, we examined the role of TLR signaling in the injury, inflammation, and repair response
in vivo. We challenged TLR2/TLR4 null mice with bleomycin and found that they had increased susceptibility
to non-infectious lung injury (Fig. 3). This was surprising since macrophages from TLR2/TLR4 null mice did
not produce inflammatory mediators in response to HA
fragment in vitro (Jiang et al., 2005). We examined the
phenotype in more detail and observed that although
mortality was increased, there was actually a decrease
in neutrophil recruitment in the absence of TLR2 and
TLR4. This presented a conundrum and clues to explain
this disconnect between an inhibition of inflammation
and an increase in tissue injury were revealed by examining the lung parenchyma in the TLR2/TLR4 null mice
after injury. Much to our surprise there was clear evidence of increased tissue damage in the TLR2/TLR4 null
Fig. 4. Overexpression of high molecular mass HA ameliorates lung
injury in CC10-HAS2 transgenic mice. CC10-HAS2 transgenic mice
have improved survival following high dose of bleomycin treatment
compared with littermate controls. Adapted from Jiang et al., 2005.
mice after injury (Jiang et al., 2005). We then examined
the lung epithelial cells and found that there was evidence of increased apoptosis in the TLR2/TLR4 null
mice after bleomycin treatment (Jiang et al., 2005).
These data suggested that somehow TLR2/TLR4 were
protective from acute lung injury due to a non-infectious
insult. We explored this further and discovered that HA
was on the cell surface of primary lung epithelial cells
from wild type mice but this cell surface expression was
significantly diminished in lung epithelial cells from
TLR2/TLR4 null mice. The implication was that cell surface HA was protective against acute lung injury. To test
this hypothesis in another way, we generated transgenic
mice that express hyaluronan synthase 2 (HAS2) specifically on lung epithelial cells using the CCSP (CC10) promoter. We found that increasing lung epithelial cell
surface HA afforded increased protection against acute
lung injury (Jiang et al., 2005) (Fig. 4). Furthermore,
when we isolated primary lung epithelial cells from
TLR2/TLR4 null mice, we found an increased susceptibility to bleomycin-induced apoptosis and high molecular
weight HA was protective against acute lung injury in a
TLR dependent manner (Jiang et al., 2005). Moreover,
Salathe and colleagues demonstrated that HA binding to
RHAMM stimulates ciliary beating to play a role in airway mucosal host defense (Forteza et al., 2001).
HA and HA receptors appear to have protean functions in lung injury inflammation and repair. These are
summarized in a schematic published as an editorial
(O’Neill, 2005) on our studies (Jiang et al., 2005). Soluble HA fragments generated during inflammation stimulate macrophages to produce mediators that are focused
on repairing the lung. However, HA on the epithelial cell
surface serves a protective function by engaging TLR2
and TLR4 to produce low levels of NF-jB activation that
are protective against exogenous insults (O’Neill, 2005).
This is analogous to the role commensal gut flora function in protecting gut epithelium form injury (RakoffNahoum et al., 2004). HA continues to be a remarkably
interesting molecule that has important roles in a variety of biological properties. HA is produced in great
abundance in the interstitium of the lung by mesenchymal cells, and it will be exciting to explore the functional
significance of this in non-infectious lung injury, inflammation, and repair.
Akira S, Takeda K. 2004. Toll-like receptor signalling. Nat Rev
Immunol 4:499–511.
Bray BA, Sampson PM, Osman M, Giandomenico A, Turino GM.
1991. Early changes in lung tissue hyaluronan (hyaluronic acid)
and hyaluronidase in bleomycin-induced alveolitis in hamsters.
Am Rev Respir Dis 143:284–288.
Forteza R, Lieb T, Aoki T, Savani RC, Conner GE, Salathe M. 2001.
Hyaluronan serves a novel role in airway mucosal host defense.
FASEB J 15:2179–2186.
Horton MR, McKee CM, Bao C, Liao F, Farber JM, Hodge-DuFour
J, Pure E, Oliver BL, Wright TM, Noble PW. 1998. Hyaluronan
fragments synergize with interferon-gamma to induce the C-X-C
chemokines mig and interferon-inducible protein-10 in mouse
macrophages. J Biol Chem 273:35088–35094.
Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R,
Lee PJ, Medzhitov R, Noble PW. 2005. Regulation of lung injury and
repair by Toll-like receptors and hyaluronan. Nat Med 11:1173–1179.
Jiang D, Liang J, Noble PW. 2007. Hyaluronan in tissue injury and
repair. Annu Rev Cell Dev Biol 23:435–461.
Medzhitov R, Janeway C, Jr. 2000. The Toll receptor family and microbial recognition. Trends Microbiol 8:452–456.
Nettelbladt O, Bergh J, Schenholm M, Tengblad A, Hallgren R. 1989.
Accumulation of hyaluronic acid in the alveolar interstitial tissue in
bleomycin-induced alveolitis. Am Rev Respir Dis 139:759–762.
Noble PW. 2002. Hyaluronan and its catabolic products in tissue
injury and repair. Matrix Biol 21:25–29.
Noble PW. 2006. Idiopathic pulmonary fibrosis: natural history and
prognosis. Clin Chest Med 27:S11–S16.
Noble PW, Homer RJ. 2004. Idiopathic pulmonary fibrosis: new
insights into pathogenesis. Clin Chest Med 25:749–758.
Noble PW, Lake FR, Henson PM, Riches DW. 1993. Hyaluronate
activation of CD44 induces insulin-like growth factor-1 expression
by a tumor necrosis factor-alpha-dependent mechanism in murine
macrophages. J Clin Invest 91:2368–2377.
Noble PW, McKee CM, Cowman M, Shin HS. 1996. Hyaluronan
fragments activate an NF-kappa B/I-kappa B alpha autoregulatory loop in murine macrophages. J Exp Med 183:2373–2378.
O’Neill LA. 2005. TLRs play good cop, bad cop in the lung. Nat Med
Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D,
Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P,
Layton B, Beutler B. 1998. Defective LPS signaling in C3H/HeJ and
C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088.
Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. 2004. Recognition of commensal microflora by Toll-like
receptors is required for intestinal homeostasis. Cell 118:229–241.
Schmits R, Filmus J, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A,
Simard JJ, Ohashi PS, Paige CJ, Gutierrez-Ramos JC, Mak TW.
1997. CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity. Blood 90:2217–2233.
Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC,
Gallo RL. 2004. Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J Biol Chem 279:17079–17084.
Taylor KR, Yamasaki K, Radek KA, Di Nardo A, Goodarzi H, Golenbock
D, Beutler B, Gallo RL. 2007. Recognition of hyaluronan released in
sterile injury involves a unique receptor complex dependent on Tolllike receptor 4, CD44, and MD-2. J Biol Chem 282:18265–18275.
Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure E, Henson
PM, Noble PW. 2002. Resolution of lung inflammation by CD44.
Science 296:155–158.
Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T,
Miyake K, Freudenberg M, Galanos C, Simon JC. 2002. Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4. J Exp Med 195:99–111.
Zaman A, Cui Z, Foley JP, Zhao H, Grimm PC, Delisser HM, Savani
RC. 2005. Expression and role of the hyaluronan receptor
RHAMM in inflammation after bleomycin injury. Am J Respir
Cell Mol Biol 33:447–454.
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hyaluronic, glycosaminoglycans, matrix, inflammation, extracellular, lung, non, injury, repair, regulation, infectious
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