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* From the University of California, Los Angeles School of Medicine, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Los Angeles, CA.
Correspondence to: Robert M. Strieter, MD, FCCP, Division of Pulmonary and Critical Care Medicine, Department of Medicine, UCLA School of Medicine, 900 Veteran Ave, 14–154 Warren Hall, Box 711922, Los Angeles, CA 90095-1922; e-mail: rstrieter{at}mednet.ucla.edu
I would first like to thank David Riches and Scott Worthen for organizing an outstanding meeting and for inviting me as conference summarizer. The presentations and discussions of mechanisms of pulmonary fibrosis at this meeting have reinforced five major issues. First, there have been and presently are outstanding investigations in the field of pulmonary fibrosis as exemplified by this conference. Second, although these studies have made outstanding contributions to further our understanding of the mechanisms related to pulmonary fibrosis, it is clear that we have the need to further our basic and clinical science knowledge of this complex process. Third, the missing link between basic science research and clinical research is that we lack information regarding the natural history of human pulmonary fibrosis, and we are left with only descriptive "snapshots" of the histopathology of human pulmonary fibrosis. This situation directly impacts on our understanding of the actual pathogenesis of these disorders. Fourth, as Dr. King points out, idiopathic pulmonary fibrosis (IPF) is a devastating disease with a 5-year survival of only 30%. This is potentially related to the fact that fibroblastic foci of granulation tissue and its potential relationship to survival highlight why our current immunosuppressive therapy is ineffective in the treatment of IPF. Fifth, based on the severity of the prognosis and the relative lack of response to currently approved medications, we need an infrastructure to support a multicenter group of investigators to facilitate the clinical study of patients with IPF. This organizational scheme would create the opportunity to study the response of these patients to novel therapeutic intervention and to establish the potential of genetic predisposition of this disorder.
In this summary, if I fail to mention the specifics of each individual’s work, I hope that these individuals will recognize the limitation of providing an all-inclusive summary of the conference, and forgive me if I do not adequately acknowledge them. The title of this conference is "Mechanisms of Pulmonary Fibrosis." The conference represented a diverse group of presentations. The talks could be generally categorized into topics related to mechanisms that perpetuate chronic inflammation and fibrosis. However, it is clear when we talk about human pulmonary fibrosis, such as usual interstitial pneumonia (UIP) associated with IPF, it is evident that we have an incomplete picture of the natural history of the pathogenesis of this disorder. The snapshot picture of UIP/IPF is a temporal nonuniform or heterogeneous pattern of alternating areas of fibroproliferation deposition of extracellular matrix (ECM), and inflammation (fibroblast foci); honeycombing of the lung parenchyma with loss of normal architecture; and normal lung tissue.1 The committee of the recent international consensus statement2 on IPF concluded that UIP is the histopathologic pattern that identifies patients with IPF, and histopathologic patterns of desquamative interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease (ILD), nonspecific interstitial pneumonia (NSIP), lymphoid interstitial pneumonia, acute interstitial pneumonia, and idiopathic bronchiolitis obliterans organizing pneumonia are considered separate entities and are to be excluded from the group of patients with IPF. However, the concern that is raised with limiting UIP to the only histopathologic description of IPF is that all of the above ILDs are idiopathic in nature and none of these disorders have a clear defined natural history of pathogenesis to allow us to fully appreciate the end point of their pathology. The reason for this is due to ethical issues, the vast majority of the patients with these disorders are treated with some form of immunosuppressive therapy, and we are unable to fully appreciate the natural history of these disorders under conditions of no treatment. To highlight this problem further, a number of participants of this meeting have personally communicated their own anecdotal experiences of having patients who initially underwent biopsy and were determined to have, for example, NSIP or bronchiolitis obliterans and organizing pneumonia, and then subsequently underwent biopsy at a later time in the natural history of the disorder and were found to have the histopathologic pattern of UIP.
Albeit anecdotal, these experiences support the notion that we do not "parachute" into the histopathologic description of UIP that is associated with IPF. There must be a continuum of dysregulated response to injury with persistence of chronic inflammation, development of granulation-like tissue, and fibrogenesis. For example, is it possible that if we do not clinically intercede with some form of therapy, that the natural history of NSIP in a number of patients could evolve into NSIP with fibrosis and ultimately into UIP? Interestingly, bronchiolitis obliterans syndrome (BOS) associated with lung transplantation is an example of a disorder of the lung that we know the natural history of the pathogenesis and clinical response to therapy. This disorder is associated with the persistent expression of an "antigen" (ie, alloantigen). The incidence of BOS is directly related to the following exacerbating and partially remitting events: episodes of acute lung allograft rejection; incidence of cytomegalovirus reactivation and pneumonitis; and bouts of Pneumocystis carinii pneumonia.3 4 What links these events to the promotion of BOS is the concept of recurrent and partially remitting chronic inflammation superimposed on the persistence of an antigen (ie, alloantigen) that ultimately leads to obliteration of the airway with granulation-like tissue and eventually fibrosis. The natural history of the pathogenesis of this disorder occurs despite aggressive intervention with potent immunosuppressive agents.4 This supports the notion that the inability of the immune system to eradicate an antigen forces the host to change strategies and simply "wall-off" the antigen using the means of fibrosis. While this form of pathogenesis may be relevant to the host, for example, to deal with a persistent antigen in the skin, the response in the lung leads to disrupted lung architecture; pathophysiology with altered lung mechanics and gas transfer; and ultimately death in 70% of lung transplant recipients at 7 years.5 This is analogous to IPF.2 Therefore, understanding the natural history of IPF will be critical to our ability to treat with the right agent at the right time of the pathogenesis of this disorder. For example, if the histopathology at a specific time is predominant chronic interstitial inflammation, it would be expected that this patient would have a high degree of likelihood to respond to conventional immunosuppressive therapy. In contrast, if the histopathology at a later stage of the natural history of the patient’s disorder demonstrated a picture compatible with UIP, then it would be highly unlikely that the patient would respond to conventional immunosuppressive therapy, and this group of patients would be the one that would benefit from a novel form of therapy to treat the underlying fibrotic phase of their disorder.
Pulmonary Fibrosis Is an Example of Dysregulated Repair and Remodeling of the Lung in Response to Injury
Normal repair following lung injury results in rapid restoration of tissue integrity and function. While normal repair is a complex interplay among humoral matrix, cellular matrix, and ECM, this process occurs in a sequential, yet overlapping, manner consisting of coagulation, inflammation, granulation tissue formation, and reestablishment of normal parenchymal-stroma cell interrelationships. In contrast to normal repair, the persistence of chronic inflammation in ILD promotes fibroproliferation and deposition of ECM reflecting dysregulated and exaggerated tissue repair. The pathogenesis of ILD is presumably related to initial loss of alveolar type-I epithelial cells and endothelial cells. However, dysregulated repair of ILD is followed by persistence of inflammation, proliferation of type-II cells, recruitment and proliferation of endothelial cells and fibroblasts, and deposition of ECM leading to end-stage alveolar and interstitial fibrosis. These events involve the complex and dynamic interplay between diverse immune effector cells and cellular constituents of the alveolar-capillary membrane and interstitium of the lung. Interaction of these diverse cell populations and the inflammatory mediators that they produce culminate in chronic inflammation and exaggerated angiogenesis, fibroproliferation, and ECM deposition.
Granulation tissue is paramount to subsequent re-epithelialization
following injury. Granulation tissue provides the foundation for
reepithelialization and reestablishment of the basement membrane.
Granulation tissue formation is associated with marked mononuclear cell
infiltration, angiogenesis, and fibroproliferation with deposition of
ECM. Under normal conditions of repair, granulation tissue formation is
temporal in nature, as there is significant involution of granulation
tissue as we have restoration of the injury. This occurs concomitantly
with reepithelialization leading to establishment of normal parenchymal
cell to stromal cell relationship. This highlights the importance of
cell-cell interaction. This is critical, as the presentations of Drs.
Worthen and Sheppard highlighted the importance of epithelial cell
interaction with fibroblasts. Dr. Sheppard showed that epithelial cells
expressing 
vß6 induce spatially restricted activation of
transforming growth factor (TGF)-ß1 that has a
direct effect on activation of fibroblasts and the generation of ECM.
This exemplifies that regulating the expression and processing of a
unique molecule on one cell can have a profound and direct paracrine
effect on a neighboring cell.
IPF is similar to other chronic fibroproliferative disorders, such as rheumatoid arthritis. The pannus of rheumatoid arthritis is analogous to granulation-like tissue, and is similar in a number of ways to the fibroblastic foci in the lungs of patients with IPF. Analogous to the pannus of rheumatoid arthritis, we have a progressive fibroproliferative process that leads to surrounding destruction of normal lung tissue. In addition, the continuum of this process may be related to the concept of exposure to "multiple hits." Multiple hits are related to recurrent events that lead to repeated exacerbations of injury with partial remissions that subsequently perpetuate chronic inflammation and fibroproliferation. For example, Dr. Tang presented compelling evidence for increased detection of herpesvirus DNA in patients with IPF, suggesting that this family of viruses may represent a persistent antigen challenge to the host. Whether herpesviruses or other microbes may be involved in the initiation of the pathogenesis of IPF remains to be determined; it is equally important to speculate that microbes may also be a mechanism that occurs after the onset of IPF, acting as a second subsequent "hit" that exacerbates the chronic inflammatory/fibroproliferative process, promotes dysregulated repair, and ultimately leads to progressive fibrosis. This scenario is not dissimilar to what we see in BOS associated with lung transplantation. Therefore, dysregulated repair in response injury in IPF may be associated with recurrent exacerbations of chronic inflammation; failure to have involution of granulation tissue with persistent angiogenesis, fibroproliferation, overexuberant deposition of ECM; and dysregulated reepithelialization that fails to reestablish parenchymal cell to stromal relationships, which is absolutely critical to retaining the normal architecture of the lung.
Initiating Events That Promote Chronic Inflammation and Fibrosis
The mediators presented at this conference can be organized into
recognition, recruitment, removal, and repair factors. In the context
of recognition, a number of presentations addressed the importance of
the early response proinflammatory factors, such as tumor necrosis
factor (TNF)-, interleukin (IL)-1, and CD40-CD40 ligand system for
their role in initiation of inflammation. Several presentations were on
the importance of leukocyte recruitment related to the expression and
function of chemokines. In addition, the biology of recruitment may be
regulated by other factors, such as metalloproteinases, that not only
regulate ECM formation, but may regulate the specific receptors on
these cell surfaces. Other investigations presented the importance of
removal factors that are involved in aiding the host in the eradication
of the inciting agent. Finally, numerous presentations addressed
factors that are involved in the resolution and remodeling phase of
repair. However, in the context of IPF and other chronic
fibroproliferative disorders, repair is dysregulated, failing to
reestablish the normal architecture of the lung.
Following tissue injury, attempts to restore tissue integrity and function are paramount and may influence the subsequent predisposition to fibrosis. Coagulation is an event that occurs early during normal repair, and is important to the development of the primordial matrix and initiation of the development of the granulation tissue response. However, dysregulated coagulation and persistent deposition of fibrin is a mechanism for promoting pulmonary fibrosis. Dr. Günther presented his work on the ability of aerosolized heparin or urokinase as novel therapeutic strategies to attenuate lung fibrosis in a rabbit model of bleomycin-induced pulmonary fibrosis. The early and late use of heparin and urokinase, respectively, markedly reduced fibrotic changes in the lungs of rabbits at 28 days as assessed by high-resolution CT of the lung.
The presence and persistence of chronic inflammation are important to
the development of pulmonary fibrosis. The expression of early response
proinflammatory factors and their persistence may be important in
mediating pulmonary fibrosis. The IL-1 family is a very important
family of cytokines. This family is one of the most complex families of
cytokines. The IL-1 family consists of two agonists (IL-1 and
IL-1ß), three alternatively spliced messenger RNAs that encode three
competitive IL-1 receptor antagonists (IL-1ra), and two receptors (type
I and II).6
One of the IL-1ra is secreted, whereas the
other two IL-1ra are intracellular and appear to be released during
cellular injury or apoptosis. In addition, the presence of two
receptors presents even further complexity, as the type-I receptor is
associated with signal coupling and the type-II receptor does not
signal and acts as a "decoy" receptor. The balance of the
expression of the agonists, antagonist, and receptors ultimately
determines the biology of IL-1. The balance of this family influences
how we can envision the role of IL-1 biology in pulmonary fibrosis.
There have been a number of studies that suggest that IL-1 promotes
fibrosis by the following: promotes inflammation through leukocyte
recruitment (ie, expression of adhesion molecules and
chemokines); induces fibroblast expression of procollagens type I and
type III, glycosaminoglycans, and fibronectin; induces fibroblast
expression of platelet-derived growth factor (PDGF); and it indirectly
mediates fibroblast proliferation.6
In contrast, there is
evidence that IL-1 may actually inhibit fibrosis. IL-1 agonists mediate
the production of tissue collagenases and prostaglandin E2
(PGE2).6
This suggests that IL-1 may be
involved in augmenting the degradation of the ECM. In addition, Dr.
Pan’s presentation demonstrated that IL-1 derived from alveolar
type-II pneumocytes inhibits fibroblast proliferation by a mechanism
related to the production of fibroblast-derived
PGE2. Moreover, a number of chronic
fibroproliferative disorders, including IPF, have been demonstrated to
have increased expression of IL-1ra in stoichiometric levels that would
inhibit the biology of IL-1 agonists.7
8
The lack of IL-1
modulation of ECM in the presence of unabated TGF-ß would lead to
heightened deposition of ECM.
Dr. Boldin’s presentation focused predominantly on TNF interaction
with one of its receptors, p55, leading to signal coupling and
apoptosis. In addition, TNF can also lead to signal coupling and
nuclear factor (NF)-
B nuclear translocation leading to
NF-B–dependent antiapoptotic and proinflammatory effects. These
disparate findings suggest that TNF may have dual and opposing effects
in regulating programmed cell death. Several presentations were made on
the importance of TNF in promoting fibrosis. Similar to IL-1, TNF can
promote pulmonary fibrosis through leukocyte recruitment
(ie, expression of adhesion molecules and chemokines) and
promotion of inflammation; induce fibroblast expression of procollagens
type I and type III, glycosaminoglycans, and fibronectin; induce
fibroblast expression of PDGF; and indirectly mediate fibroblast
proliferation. In contrast, there is evidence that TNF may inhibit
fibrosis by enhancing the production of fibroblast collagenases and
PGE2. Nevertheless, Dr. Ortiz presented that TNF
receptor -/- mice are protected from silica-induced pulmonary fibrosis
by altering lung interstitial collagenase (matrix metalloproteinase
[MMP]-13) and tissue inhibitor of metalloproteinase (TIMP)-1
expression. Under these conditions, ECM undergoes more rapid
degradation by reduced levels of TIMP-1 expression. This presentation
supported the findings of other studies that demonstrated transgenic
overexpression of TNF in the lung results in progressive pulmonary
fibrosis.9
While TNF and IL-1 are important early response cytokines that are
involved in fibroblast activation, Dr. Phipps’ presentation
highlighted the importance of CD40 signaling in fibroblasts. Lung
fibroblasts express the receptor, CD40, and when stimulated by CD40
ligand (CD154) derived from lymphocytes, platelets, and mast cells,
fibroblasts are activated to synthesize a variety of factors, such as
PGE2, IL-6, and IL-8, and extracellular proteins
(hyaluronate). This activation is similar to activation by IL-1 and TNF
and appears to be mediated through NF-B nuclear translocation and
the transactivation of the promoter of these genes. These findings for
the importance of CD40-CD40 ligand system together with TNF and
IL-1 may be essential elements in initiating and perpetuating chronic
pulmonary inflammation and fibrosis.
Maintenance of Inflammation: the Recruitment Cytokines, Chemokines
Although not all inflammatory disorders result in fibrosis, fibrotic responses are always preceded and potentially perpetuated by chronic inflammation. The salient feature of chronic inflammation is the association of mononuclear cell infiltration. These extravasating leukocytes contribute to the pathogenesis of chronic inflammation and promote fibrosis via the elaboration of a variety of cytokines. The maintenance of leukocyte recruitment during inflammation requires intercellular communication between infiltrating leukocytes and the endothelium, resident stromal, and parenchyma cells. These events are mediated via the generation of early response proinflammatory factors, eg, IL-1, TNF, and CD40-CD40 ligand; the expression of cell-surface adhesion molecules; and the production of chemotactic molecules, such as chemokines.
The recruitment of mononuclear cells and perpetuation of chronic inflammation appear to be important in promoting pulmonary fibrosis. The CC chemokine family is an especially important chemokine family, as most members of the of the CC chemokine family cause recruitment of mononuclear cells.10 Monocyte chemoattractant protein (MCP)-1 has been found to be markedly elevated in the lungs of patients with IPF, and attenuation of its expression during bleomycin-induced pulmonary fibrosis is associated with marked reduction in total lung collagen.11 12 The mechanism for this effect is the attenuation of the recruitment of mononuclear phagocytes, activation, and perhaps elaboration of additional macrophage-derived profibrotic factors. Two presentations at this conference demonstrated the importance of MCP-1 and its receptor, CCR2, in promoting fibrosis. Dr. Belperio’s presentation focused on the role of MCP-1/CCR2 in mediating the fibro-obliteration of the airway associated with bronchiolitis obliterans/chronic lung allograft rejection. The effect of MCP-1/CCR2 in this model system using CCR2 -/- mice appeared to be mediated through the recruitment of a profibrotic mononuclear phagocyte, and was independent of the recruitment of CD4+ and CD8+ T cells. In another presentation, Dr. Moore demonstrated that MCP-1/CCR2 biology played an important role in mediating pulmonary fibrosis in a model system of fluorescein isothiocyanate-induced pulmonary fibrosis. CCR2 -/- mice were less susceptible to pulmonary fibrosis, as compared to +/+ mice.
While chemokines are important in recruiting a variety of leukocytes,
it is increasingly clear that chemokine biology is expanding. There is
evidence that nonleukocyte populations of cells (ie,
epithelial cells, fibroblasts, and endothelial cells) express chemokine
receptors. MCP-1 interaction with CCR2 on fibroblasts has been
previously demonstrated to be important for autocrine and paracrine
induction of fibroblast procollagens, via the expression of TGF-ß.
This event may be more important depending on the environment in which
the fibroblast resides. Dr. Kunkel’s presentation demonstrated that
MCP-1 is involved in fibrosis through the regulation of profibrotic
fibroblast-derived cytokine generation and matrix
deposition.13
Changes in MCP-1 and its receptor CCR2,
procollagen I and III, and TGF-ß were examined in fibroblasts
cultured from normal lung and from nonfibrotic (ie, T-helper
[Th]1-type) and fibrotic (ie, Th2-type) pulmonary
granulomas. Fibroblasts isolated from Th2 lesions generated twofold
more MCP-1 than similar numbers of fibroblasts from Th1-type lesions or
normal fibroblasts. Th2-type fibroblasts expressed CCR2 messenger RNA
within 24 h after IL-4 treatment. In addition, IL-4 stimulation
also increased the number of normal fibroblasts expressing cell-surface
CCR2. In contrast, treatment with interferon (IFN)-
significantly
decreased the numbers of CCR2 expressing normal and Th2-type
fibroblasts. CCR2 expression was functional, as MCP-1 induced the
expression of TGF-ß and type-I and type-III procollagens. These
findings suggest that lung fibroblasts can express CCR2 and respond to
MCP-1 leading to a more profibrotic fibroblast phenotype. The above
presentations support the notion that that chemokines, especially
MCP-1, are pleiotropic in their biological effects during the
pathogenesis of pulmonary fibrosis.
Resolution of Inflammation and Removal Factors
The resolution of inflammation after injury is a critical event that leads to normal resolution and repair. Mechanisms that appear to be important to resolution are related, in part, to the ability of inflammatory cells such as neutrophils to undergo apoptosis and be recognized and removed by macrophages. Dr. Haslett’s presentation highlighted the importance of apoptosis in the resolution of lung inflammation. His presentation focused on apoptosis of the neutrophil and the critical ability of the macrophage to phagocytize the apoptotic neutrophils in order to effectively clear these cells after an inflammatory event. Dysregulated clearance of these cells may impact in a variety of ways that lead to perpetuation of the inflammatory response. Abnormalities in recognition and phagocytosis of apoptotic neutrophils by macrophages may lead to the expression of proinflammatory molecules by the macrophage. CD44 is a glycoprotein expressed on the cell surface of macrophages, and is the major receptor for the glycosaminoglycan hyaluronan. CD44 expression and recognition of hyaluronan on apoptotic cells enhance the clearance of apoptotic cells. Dr. Noble presented that CD44 is required for resolution of lung inflammation during bleomycin-induced pulmonary fibrosis. In CD44 -/- mice, bleomycin induced intense inflammation that was correlated to the persistence of apoptotic neutrophils, expression of proinflammatory/fibrotic genes, and mortality. These findings support the notion that removal of inflammatory cells is critical to resolution, and failure to clear these cells is associated with the persistence of inflammation and the release of proinflammatory and fibrotic genes that may be relevant to promoting pulmonary fibrosis.
While clearance of apoptotic inflammatory cells is critical to
resolution of inflammation, the removal of an inciting agent, such as a
microbe, is also essential for resolution of inflammation. Dr.
Whisett’s presentation highlighted the importance of surfactant
protein A and surfactant protein D as collectins in mediating the
innate host response, and their importance in removal of bacteria and
viruses. Knockout model systems of these collectins demonstrate
increased susceptibility to viral and bacterial infection. In fact,
surfactant protein D -/- mice demonstrate a predisposition to pulmonary
fibrosis. In another presentation related to metalloproteinases, Dr.
Parks presented information related to the critical role that
epithelial cell-derived MMP-7 plays in processing and ß
defensins. The critical nature of this system is related to whether
MMP-7 is apical or basolateral secreted from the epithelial cells.
Defensins require activation by cleavage of their precursor peptide in
order to achieve microbicidal activity. When apically secreted, MMP-7
is able to process defensins for microbicidal activity. However, when
the epithelium is injured, MMP-7 is secreted in a basolateral manner.
This effect spatially and functionally changes the substrate for MMP-7.
The result is a loss of the ability of MMP-7 to process defensins on
the apical surface of the epithelium leading to reduced microbicidal
activity, and concomitantly results in an increase in basolateral
secretion of MMP-7 and degradation of matrix constituents and matrix
remodeling. This situation may be relevant to IPF. For example, one
could speculate that if the epithelium is injured by a antigen/microbe
and removal of this same antigen/microbe is defensin dependent, the
following scenario would occur: (1) MMP-7 would not be apically
secreted leading to impaired processing of defensins and persistence of
the microbe/antigen; (2) MMP-7 would be basolaterally secreted, leading
to increased matrix remodeling; and (3) the combined effect of (1) and
(2) would lead to persistence of the microbe/antigen contributing to
"multiple hits," promotion of chronic inflammation, and ultimately
pulmonary fibrosis. These findings suggest that if problems exist
within the innate host response with failure to remove a
microbe/antigen, the persistence of the microbe/antigen may be a means
to perpetuate chronic inflammation and promote fibrosis. This fits with
the concept of multiple hits related to the vicious cycle of
exacerbations and partial remissions that ultimately leads to
dysregulated repair/remodeling and end-stage fibrosis.
Th1 and Th2 Paradigm of Pulmonary Fibrosis
Dr. Kunkel’s presentation highlighted evidence supporting the
paradigm that the initiation, maintenance, and resolution of an immune
response is governed by a complex network of specific cytokines. The
discovery that Th-cell subsets could be classified on the basis of
cytokine profiles has provided a degree of clarification to chronic
cell-mediated immune responses. The type-1 (Th1) and type-2 (Th2)
cytokines include IL-18, IL-12, IFN-, and IL-2 vs IL-4, IL-5, IL-10,
and IL-13, respectively.14
The realization that Th1 and Th2 cytokines are expressed by a variety of cells and the functions of these cytokines are different suggests that an imbalance in the expression of Th1 and Th2 cytokines may be important in dictating different immunopathologic responses.14 15 16 For example, type-1 cytokines appear to be involved in cell-mediated immunity associated with autoimmune disorders and acute allograft rejection, whereas type-2 cytokines are predominantly involved in mediating allergic inflammation and chronic fibroproliferative disorders, such as asthma, atopic dermatitis, IPF, and systemic sclerosis. Thus, it is more appropriate to define certain diseases in terms of the predominant cytokine profile rather than the predominate Th-cell subset. The strict definition of Th1 and Th2 responses may break down in a scenario where the initial inciting agent triggers an unsuccessful Th1-type response. The subsequent host reaction to a specific antigen or the chronicity of the disorder may induce a switch to a response dominated by Th2 cytokines. The manifestation of this latter response is the scenario of stromal cell/fibroblast proliferation and deposition of ECM, and ultimately fibrosis. Thus, the cytokine pattern in particular diseases is often predictable and appropriate, whereas severe pathologic consequences may result if an inappropriate cytokine phenotype is expressed. This latter situation may play a role in certain chronic inflammatory diseases, such as pulmonary fibrosis, where unknown etiologies lead to dysregulated repair with exaggerated chronic inflammation, fibroblast proliferation, deposition of ECM, angiogenesis, and finally end-stage pulmonary fibrosis.
There is evidence suggesting that a cytokine profile of the
immune/inflammatory response determines the disease phenotype
responsible for either resolution or progression to end-stage fibrosis.
Supporting evidence is derived from studies demonstrating that IFNs,
especially IFN-, have profound suppressive effects on the production
of ECM proteins, such as collagen and fibronectin. Investigations have
demonstrated that IFN- can inhibit both fibroblast and chondrocyte
collagen production in vitro, as well as decrease the
expression of steady-state type-I and type-III procollagen messenger
RNA. IFN- reduces PDGF-induced lung fibroblast growth but stimulates
PDGF production by alveolar macrophages. IFN- upregulates the major
matrix-degrading metalloproteinase, stromelysin-1 gene expression by
fibroblasts. IFN- is a potent inhibitor of the eosinophil
chemotactic CC chemokine, eotaxin from fibroblasts. IFN-
differentially regulates intracellular adhesion molecule-1 and vascular
cell adhesion molecule-1 expression on fibroblasts. The administration
of IFN- in vivo can cause a reduction of ECM in animal
models of fibrosis. Moreover, IFN- treatment of patients with either
systemic sclerosis or IPF for 1 year has demonstrated improved
pulmonary function and gas exchange with improved resting and exercise
PaO2. This information
supports the concept that IFN- is one of the major type-1 cytokines
that possesses profound regulatory activity for collagen deposition
during chronic inflammation.
While other type-1 cytokines have not been fully evaluated for their
role modulating pulmonary fibrosis, IL-18 and IL-12 are cytokines that
are important in the induction of IFN-. Dr. Keane presented evidence
that treatment with IL-12 attenuates pulmonary fibrosis by an
IFN-–dependent mechanism. The administration of IL-12 to mice
exposed to bleomycin-induced pulmonary fibrosis resulted in a
time-dependent expression of IFN- that paralleled the reduction in
pulmonary fibrosis. The antifibrotic effect of IL-12 could be entirely
attenuated when animals were passively immunized with neutralizing
antibodies to IFN-. In contrast to the above findings for IFN- in
pulmonary fibrosis, Dr. Chen presented that IFN- -/- mice display
markedly reduced inflammation and subsequent development of pulmonary
fibrosis in response to bleomycin. The disparity in the results of this
study and all the other studies implicating IFN- as an inhibitor of
fibrosis may be simply related to temporal events related to the
continuum of inflammation and fibrosis that is often seen in the
bleomycin-induced pulmonary fibrosis model system. Dr. Chen’s model
system also highlighted the concept of no inflammation no fibrosis. In
contrast, while IFN- may promote inflammation early in the
bleomycin-induced pulmonary fibrosis, the persistence of its expression
either endogenously or administered exogenously is important to
attenuate fibrosis.
The opposing effects of Th1 and Th2 cytokines in fibrosis are further
supported by a number of recent investigations17
18
19
demonstrating that IL-4 is an important mediator of fibroblast
activation. In contrast to IFN-, IL-4 is a major Th2-type cytokine
that promotes the production of fibroblast-derived ECM, including
type-I and type-III procollagens and
fibronectin.17
18
19
IL-4 treatment of fibroblasts
leads to increases in steady-state levels of ECM messenger RNA and
protein. IL-4 has been identified as a chemotactic factor for
fibroblasts.20
IL-4 can induce fibroblast proliferation
and cytokine production. Interestingly, the intensity of IL-4–induced
fibroblast collagen synthesis is on the same order of magnitude as that
induced by equal amounts of TGF-ß. The above information underscores
the pleiotropic activities of IL-4, as fibroblast activation must be
included on the list of activities along with the proliferation of B
cells, T lymphocytes, mast cells, and hematopoietic progenitor cells.
IL-13 has similar biological properties to IL-4, and has been implicated in the pathogenesis of fibroproliferative disorders.21 IL-13 induces the expression of fibroblast-derived type-I and type-III procollagens in a similar magnitude as IL-4 and TGF-ß.21 IL-13 inhibits IL-1–induced MMP-1 and MMP-3 production, and enhances TIMP-1 generation from fibroblasts.21 Recently, IL-13 was selectively expressed in the lungs using a Clara cell promoter.22 The phenotype of the transgenic mice expressing IL-13 demonstrated airway epithelial cell hypertrophy, mucous cell metaplasia, the hyperproduction of neutral and acidic mucus, the deposition of Charcot-Leyden–like crystals, and subepithelial airway fibrosis.22 These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.
Lung tissue of patients with IPF has been examined for the presence of
a Th1 vs Th2 pattern of cytokine expression by in situ
hybridization and immunolocalization of cytokine
protein.23
Although there is a pattern for the existence
of both Th1 (characterized by the expression of IFN-) and Th2
(characterized by the expression of IL-4 and IL-5 cytokines in IPF lung
tissue), the presence of Th2 cytokines predominated over the expression
of IFN-.23
This pattern of cytokine expression may be
related to the potential role for the humoral response in the
pathogenesis of IPF or be related to the inability of IFN- to tilt
the balance away from an IL-4/IL-13–dependent profibrotic environment.
In further support of an imbalance of the presence of Th2 cytokines as
compared to IFN- is the finding that IFN- levels are inversely
related to the levels of type-III procollagen in the BAL fluid (BALF)
of IPF patients.24
The levels of IFN- were especially
correlated with patients who demonstrate progression of their pulmonary
fibrosis by evidence of further deterioration of their pulmonary
function.24
These findings have been further substantiated
with the recent finding that IPF patients who had failed to
respond to conventional immunosuppressive therapy and were treated with
IFN- for 1 year responded with improved total lung capacity and
resting and exertional
PaO2.25
The
predominance of Th2, as compared to Th1 cytokines in chronic
inflammation/fibroproliferation of IPF supports the notion that these
removal cytokines are probably inadequate to fully eliminate the
inciting antigen, and the promotion of fibrosis by Th2 cytokines may be
an inept attempt to contain or wall off the antigen. These findings
suggest that the persistent imbalance in the expression of Th2 vs Th1
cytokines in the lung is a mechanism for the progression of pulmonary
fibrosis.
Extracelluar Matrix Remodeling, Fibrosis, and Angiogenesis
Extracellular matrix formation and remodeling are critical events related to the pathogenesis of pulmonary fibrosis. MMPs play an essential role in degrading and remodeling the ECM. Dr. Werb presented an overview of MMPs and presented their importance in mediating branching morphogenesis of mammary glands in mammary fat pads. She demonstrated the importance of MMPs in regulating stromal cell to parenchymal cell interaction. Interestingly, using genetic approaches, MMP-3/stromelysin-1 overexpression using the whey acidic protein gene promoter resulted in a marked increase in mammary gland fibrosis in transgenic mice. These changes were absent in wild-type littermates and were quenched by coexpression of a human TIMP-1 transgene. In contrast, serine proteinases, such as neutrophil elastase, have the opposite effect leading to reduced fibrosis in her model system.
Increased expression of MMPs, such as MMP-9/gelatinase B, has been demonstrated in the lungs of patients with IPF. To determine the role of MMP-9 in pulmonary fibrosis, Dr. Betsuyaku, using MMP-9 -/- mice, demonstrated that MMP-9 is required for alveolar bronchiolization with Clara cell features, but not fibrosis during the pathogenesis of bleomycin-induced lung fibrosis. Dr. Fukuda’s presentation highlighted that in UIP of IPF patients, alveolar structures are lost and replaced by bronchiolization with smooth-muscle hyperplasia. Dr. Roman presented that increased gelatinolytic activity was seen in the BAL of sarcoidosis patients, and the activity was directly correlated with radiographic staging and impaired pulmonary function. These findings suggested that MMPs are important in facilitating reepithelialization in an attempt to reestablish bronchiolization after alveolar injury and may be important to remodeling the ECM during disorders associated with pulmonary fibrosis.
Fibroblast proliferation is an essential event in pulmonary fibrosis. It is increasingly clear that activation of receptor tyrosine kinases and the subsequent activation of mitogen-activated protein (MAP) kinase pathways are important in initiating the molecular signaling events that mediate cellular proliferation necessary for fibroblast mitogenesis. Dr. Bonner’s presentation highlighted the importance of PDGF receptor and epidermal growth factor receptor in mediating fibroblast proliferation via receptor tyrosine kinase activation of the MAP kinases, extracellular signal-regulated kinases 1 and 2, and p38 MAP kinase during the pathogenesis of vanadium pentoxide-induced pulmonary fibrosis. In addition, by specifically attenuating the tyrosine kinase pathway, there was a marked reduction of pulmonary fibrosis, suggesting that therapeutic intervention and disruption of this signal transduction pathway may offer novel therapeutic intervention to treat pulmonary fibrosis.
While fibroblast proliferation is important in the pathogenesis
of fibrosis, the expression and synthesis of fibroblast-derived ECM, as
compared to its degradation, are critical to the promotion of fibrosis.
TGF-ß is the most potent promoter of fibrosis. TGF-ß belongs to a
superfamily of genes and exists as three closely homologous (72 to
80%) dimeric isoforms, TGF-ß1,
TGF-ß2, and
TGF-ß3.26
27
28
The three isoforms
of TGF-ß are initially translated as a prepropolypeptide that
contains the hydrophobic signal sequence, a latency-associated peptide,
and the mature form of the monomeric TGF-ß. TGF-ß undergoes initial
NH2-terminal cleavage with the removal of a 29
amino-acid signal peptide, followed by dimerization due to the
formation of a disulfide bond, and then it is secreted as an inactive
latent TGF-ß complex. Destabilization of the latent TGF-ß complex
and release of the active 25-kd cytokine occur with either proteolytic
activation or alterations in the ionic environment, eg, an
acidic pH. While the extracellular activation of these complexes is a
critical but incompletely understood step in the regulation of TGF-ß
function in vivo, Dr. Sheppard presented that
TGF-ß1 latency-associated peptide is a ligand
for the integrin vß6 and that vß6-expressing epithelial cells
induce spatially restricted activation of
TGF-ß1. The importance of this biology was
demonstrated in mice that were vß6 -/- and exposed to bleomycin
and were found to be protected from pulmonary fibrosis. These data
identify a novel mechanism for locally regulating
TGF-ß1 function in vivo by
regulating expression of the vß6 integrin. In addition,
these data highlight the importance of cell-to-cell interaction with
the ability of an vß6 expressing cell, such as an epithelial cell,
to spatially communicate with a fibroblast with presentation of active
TGF-ß1.
The TGF-ß receptors (types I and II) are serine-threonine kinase
receptors. The use of yeast two hybrid system for determining
protein-protein interaction has identified important components of the
TGF-ß signal transduction system. This understanding of the TGF-ß
signal transduction pathway has allowed studies to be designed to
target specific proteins in the pathway to specifically determine their
precise role in mediating TGF-ß response in vivo. Dr.
Roberts’ presentation highlighted the importance of a TGF-ß
receptor-activated family of latent transcription factors called
"Smads" in mediating signal transduction of TGF-ß. She showed
that a set of receptor-activated Smads are phosphorylated directly by
the receptor kinase and then translocate to the nucleus complexed to
the common mediator, Smad-4, to participate in transcriptional
complexes. Both Smad-3 and its closely related homolog Smad-2 act as
latent nuclear transcriptional activators and mediate intracellular
signaling by TGF-ß. In contrast, IFN- can inhibit TGF-ß
signaling by induction of the inhibitory Smad, Smad-7, which blocks the
actions of Smad-3. These data implicate Smad-3 in specific pathways of
tissue repair and fibrosis, and may suggest a potential target for the
development of therapeutic strategies to modulate pulmonary fibrosis.
TGF-ß has been found by immunohistochemistry and is localized to bronchiolar epithelial cells, epithelial cells of honeycomb cysts, and hyperplastic type-II pneumocytes in the lungs of IPF patients. In addition, TGF-ß has been found in IPF in association with constituents of the ECM. The predominant isoform of TGF-ß in IPF, similar to what has been found in bleomycin-induced pulmonary fibrosis, is TGF-ß1. TGF-ß protein levels from lung tissue homogenates of IPF patients have been measured by specific enzyme-linked immunosorbent assay and found to be 11-fold higher, as compared to levels in normal control subjects. Interestingly, no significant difference in the levels of TGF-ß has been found in patients who exhibited a predominant histopathologic pattern of desquamative interstitial pneumonitis vs UIP. In addition, levels of TGF-ß in BAL from IPF patients may directly correlate with mortality. To determine whether TGF-ß is a predominant factor for the promotion of pulmonary fibrosis, studies using bleomycin-induced lung fibrosis have demonstrated that TGF-ß plays an important role in this process. To further confirm this biology, Dr. Brody demonstrated that in mice less susceptible to bleomycin-induced pulmonary fibrosis, reconstitution of TGF-ß using an adenoviral vector delivery system of the TGF-ß gene resulted in increased inflammation and a marked fibroproliferative response. These findings suggest that TGF-ß is a critical cytokine for the promotion of pulmonary fibrosis.
Angiogenesis is a fundamental component of inflammation and wound repair that is relevant to pulmonary fibrosis. However, it has only recently become apparent that dysregulated angiogenesis may be important in the support of fibroplasia and deposition of ECM that occurs during chronic fibroproliferative disorders, such as IPF. The existence of neovascularization in IPF was originally identified by Turner-Warwick, who examined the lungs of patients with widespread interstitial fibrosis and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculatures and evidence of extensive vascular remodeling in areas of fibrosis. These findings have been further substantiated with evidence of extensive neovascularization during the pathogenesis of pulmonary fibrosis in a rat model of bleomycin-induced pulmonary fibrosis.
Our laboratory has shown that angiogenesis in pulmonary fibrosis is
related the overexpression of ELR-positive CXC chemokines, as compared
to angiostatic (ELR-negative) IFN-inducible CXC chemokines. BALF and
lung tissue from patients with IPF have been demonstrated to have
markedly increased angiogenic activity that is almost entirely
attributable to the imbalance in the overexpression of the angiogenic
ELR-positive CXC chemokine, IL-8, as compared to the relative
downregulation of the angiostatic IFN-inducible CXC chemokine, IP-10.
To determine whether the imbalance in the expression of these CXC
chemokines is relevant to the pathogenesis of pulmonary fibrosis, the
expression and biological activity of murine macrophage inflammatory
protein (MIP)-2 (an angiogenic ELR-positive CXC chemokine homologous to
human GRO-/ß) and the angiostatic CXC chemokine, IP-10, were
correlated with the extent of fibrosis during bleomycin-induced
pulmonary fibrosis in a murine model system. MIP-2 and IP-10 were
temporally measured during bleomycin-induced pulmonary fibrosis from
BAL and whole-lung tissues, and were found to be directly and inversely
correlated, respectively, with total lung hydroxyproline levels, a
measure of lung collagen deposition. Moreover, if either
endogenous MIP-2 was depleted by passive immunization with neutralizing
antibodies, or exogenous IP-10 was administered to the animals during
bleomycin exposure, both treatment strategies resulted in marked
attenuation of pulmonary fibrosis that was entirely attributable to a
reduction in angiogenesis in the lung. These findings support the
notion that angiogenesis is a critical biological event that supports
fibroplasia and deposition of ECM in the lung during pulmonary
fibrosis, and that angiogenic and angiostatic factors, such as CXC
chemokines, play an important role in the pathogenesis of this process.
Furthermore, as to the demonstration of the efficacy of IFN-
treatment of patients with IPF, the above studies substantiate that
IFN- treatment of IPF may mediate its effect, in part, by shifting
the imbalance of the expression of ELR-positive and ELR-negative CXC
chemokines to favor an angiostatic environment leading to inhibition of
dysregulated neovascularization/vascular remodeling,
fibroproliferation, and deposition of ECM in IPF patients.
I have attempted to summarize the putative role of a variety of factors
that were presented at this conference that may contribute to pulmonary
fibrosis. I have categorized these factors on their ability to function
in recognition, recruitment, removal, and repair/remodel in the context
of the chronic inflammatory/fibroproliferative nature of pulmonary
fibrosis. Interestingly, many of these factors are detectable in the
BALF or lung tissue of patients suffering pulmonary fibrosis and may
serve as useful laboratory markers of disease activity or novel targets
for therapeutic intervention. Given the sometimes limited efficacy of
the therapeutic armamentarium currently used to treat ILD/IPF patients,
it seems prudent to consider alternative novel therapeutic regimens,
such as ablation of multiple factors, in order to prevent pulmonary
fibrosis and the associated pathophysiology. Dr. Hunninghake’s
presentation highlighted a variety of potential targets in regard to
novel intervention. The issue related to the natural history of IPF is
important as to when, what, and where to treat in order to maximally
impact on the disorder with current or novel therapy. The need for an
infrastructure and multicenter approach to the treatment of
patients suffering from pulmonary fibrosis is obvious due to concerns
of confounding variables and the need to perform prospective and
blinded studies on the right population of ILD patients. The recent
discovery that IFN- treatment of IPF patients results in improved
lung function is exciting and suggests that the administration of a
pivotal cytokine, IFN-, may have a dramatic impact on a variety of
other factors that ultimately reduce pulmonary fibrosis. Future
directions in clinical research may include systemic or local
intrapulmonary factor gene, protein, or small molecule therapy, which
may either attenuate or augment the expression of specific
proinflammatory/profibroproliferative factors. Hopefully, the study of
these mediators will lead to more specific therapies that will benefit
patients with pulmonary fibrosis, and prevent end-stage pulmonary
fibrosis leading to reduced morbidity and mortality.
Footnotes
Abbreviations: BALF = BAL fluid; BOS = bronchiolitis obliterans syndrome; ECM = extracellular matrix; IFN = interferon; IL = interleukin; IL-1ra = interleukin-1 receptor antagonist; ILD = interstitial lung disease; IPF = idiopathic pulmonary fibrosis; MAP = mitogen-activated protein; MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; MMP = matrix metalloproteinase; NF = nuclear factor; NSIP = nonspecific interstitial pneumonia; PDGF = platelet-derived growth factor; PGE2 = prostaglandin E2; Th = T-helper; TGF = transforming growth factor; TIMP = tissue inhibitor of metalloproteinase; TNF = tumor necrosis factor; UIP = usual interstitial pneumonia
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