Abstract
Biological therapies for the management of immune mediated inflammatory diseases such as rheumatoid arthritis have proven to be extremely successful in recent years. Despite these successes, even the most effective of therapies do not lead to cure. Why chronic inflammation persists indefinitely within the rheumatoid synovium despite an absence of continuous stimulation, and why some patients with early synovitis progress to persistent disease whilst others do not, has remained unexplained. In contrast to the paradigm that stromal cells are biochemically active but immunologically passive, there is now growing evidence that stromal components from the rheumatoid synovium play a crucial part in the immunopathology of rheumatoid arthritis. Stromal cells play a central role in the transformation of an acute, resolving to a chronic inflammatory process, and to the persistence of synovial inflammation and joint destruction through a variety of immune mechanisms. Therapeutic manipulation of the stroma is a largely unexplored, yet potentially vital area of research. Targeting pathogenic stromal cells has the potential to provide a cure for chronic inflammatory disorders such as rheumatoid arthritis.
Introduction
Inflammation is an intrinsic, biological response that occurs to protect organisms against harmful stimuli. At a cellular level, inflammation is characterised by an influx of leukcocytes into damaged or infected tissue. The duration, type and magnitude of leucocyte influx is controlled by cytokine and chemokine gradients. For an inflammatory response to resolve completely, these gradients must be removed, and leucocytes must be cleared from the site of pathology, either by migration out of the site, or via apoptosis .
Episodes of acute inflammation most often occur in the context of infection; these episodes are transient, often self-limiting and are followed by a phase of tissue repair leading to health. Chronic, persistent inflammation in contrast often occurs in the absence of a pathogen, and in the case of autoimmune diseases such as rheumatoid arthritis, may persist indefinitely even in the absence of a harmful external stimuli.
The current and established pharmacological management of most autoimmune pathologies aims to dampen the persistent, harmful inflammatory response in the tissue affected. This approach of minimising damage and progression of disease is however suboptimal. The optimal approach is to reverse the fundamental pathological process.
Our understanding of the role that stromal cells play in inflammation has changed considerably. Stromal cells, in particular fibroblasts, are emerging as key drivers in the genesis of tissue specific inflammation, modulation of tissue microenvironments, transformation from acute to chronic inflammation and persistence of inflammation through a multitude of immune-mediated modalities, particularly in rheumatoid arthritis . These discoveries raise the possibility that stromal cells may turn out to be novel therapeutic targets in the discovery of a cure for chronic inflammatory diseases.
This review aims to highlight the role that fibroblasts, a key cellular component of the stroma, play in the genesis and persistence of chronic inflammation in rheumatoid arthritis and to provide an insight into how currently available biologic agents modulate stromal cell function. It will also outline possibilities for novel stromal cell targets, which may be beneficial in future therapies for rheumatoid arthritis.
The stroma
Tissue stroma comprises the subendothelial basement membrane and the underlying connective tissue containing stromal cells such as pericytes, fibroblasts and organ specific stromal cells (astrocytes, hepatocytes) and epithelial cells as well as adipocytes . The extracellular matrix that lies between stromal cells is comprised mainly of gylcosaminoglycans and proteoglycans. Lymphatic and blood vessels and nervous tissue is interspersed throughout the stroma. It is through this matrix that leucocytes migrate to and from an area of inflammation . Considerable effort has been expended in targeting vascular endothelial cells in cancer and inflammation. While new anti-angiogenic agents have shown promise in some cancers, their application to autoimmune diseases remain disappointing.
Fibroblast biology
The fibroblast is the most common cell type within the stroma . Fibroblasts are identified by their spindle-shape morphology, expression of interstitial collagens (types I and III) and their adherence to plastic in culture . Fibroblasts express numerous markers which aid in their identification ( Table 1 ). However None of these markers is highly specific to fibroblasts, nor is any marker reliably present in all fibroblasts . Because of this, there has been until recently a lack of information on the origin, function and role of fibroblasts in disease .
| Marker | Function | Fibroblast subtype | Expressed elsewhere |
|---|---|---|---|
| Vimentin | Intermediate filament associated protein | Miscellaneous | Endothelial cells, myoepithelial cells, neurons |
| α-SMA | Intermediate filament associated protein | Myofibroblasts | Vascular smooth muscle cells, pericytes, myoepithalial cells |
| Desmin | Intermediate filament associated protein | Skin fibroblasts | Muscle cells, vascular smooth muscle cells |
| FSP1 | Intermediate filament associated protein | Miscellaneous | Invasive carcinoma cells |
| Discoidin-domain receptor 2 | Collagen receptor | Cardiac fibroblasts | Endothelial cells |
| FAP* | Serine protease | Activated fibroblasts | Activated melanocytes |
| α1β1 integrin | Collagen receptor | Miscellaneous | Monocytes, endothelial cells |
| Prolyl 4-hydroxylase | Collagen biosynthesis | Miscellaneous | Endothelial cells, cancer cells, epithelial cells |
| Pro-collagen-1α2 | Collagen-1 biosynthesis | Miscellaneous | Osteoblasts, chondroblasts |
| CD248* | Unknown | Miscellaneous | Pericytes |
| VCAM-1 | Cell adhesion | Miscellaneous | Activated endothelial cells |
| gp38* | Invasiveness in cancer | Miscellaneous | Alveolar cells, podocytes, endothelial cells, neurons |
| Cadherin-11* | Homotypic adhesion | Miscellaneous | Nil |
| Recruitment | TLO formation | Retention | Reduced emigration | Reduced apoptosis |
|---|---|---|---|---|
| IL-1 | gp38 | IFN-1 | CXCL12 (SDF-1) | Bcl-x L |
| IL-6 | CD157 (BP-3) | IL-15 | CCL21 | BCl-2 a |
| TNFα | IL-7 | BAFF | IFN-β | |
| COX-2 | CXCL13 (BLC) | CXCL12 (SDF-1) | NF-κB | |
| CXCL8/IL-8 | CCL19 | CXCR4 | IL-2 | |
| CCL5 | CCL21 (SLC) | TGF-β | IL-4 | |
| CXCL1 | ICAM-1 | IL-15 | ||
| CXCR2 | VCAM-1 | BAFF b | ||
| CXCL5 | APRIL b | |||
| CXCL12 (SDF-1) | IL-6 b | |||
| CXCL12 b (SDF-1) |
a Low levels of BCl-2 are observed during fibroblast mediated inhibition of lymphocyte apoptosis. All other molecules are upregulated by fibroblasts to modulate persisting inflammation.
Mesenchymal Stromal cells (MSCs) are multipotent cells, which are currently undergoing extensive investigation for use in tissue repair. They share common cell surface markers, morphology and immunologic properties with fibroblasts . It is currently, although not exclusively accepted, that MSCs and fibroblasts are distinct cell types, set apart by the ability of MSCs to differentiate into any mesodermal tissue and the discovery that fibroblasts are not always mesodermal in origin. For example fibroblasts in the head and neck arise from the ectoderm through a process termed epithelial to mesenchymal transition (EMT) .
Under physiological conditions, the stroma functions primarily to provide structure by synthesising and remodelling the extracellular matrix (ECM). Fibroblasts synthesise type I, type III and type V collagen, and fibronectin but also have a role ECM degradation via matrix metalloproteinases (MMPs) . Thus fibroblasts have prime responsibility for the maintenance of ECM homoeostasis by regulating matrix turnover . Fibroblasts also help in maintaining biochemical homoeostasis between adjacent epithelial cells and have a key role in tissue development, differentiation and repair (including fibrosis) .
Of the cells that comprise the stroma, the fibroblast has been most implicated in generating and directing both the innate and adaptive immune response in health and disease . Synovial fibroblasts from patients with rheumatoid arthritis have provided key information on how the stroma is responsible for persistence of chronic inflammation. This is discussed in detail below.
Pathogenic stroma
Stromal tissue has been shown to play a functional role in three chronic pathological states: malignant disease, tissue fibrosis and chronic inflammation . In all of these conditions, stromal cells have been observed to undergo specific, disease dependent phenotypic and functional changes which promote the disease state . In malignancy for example, in addition to production of the non-tumour extracellular matrix that facilitates growth of solid tumours, the stroma has been shown to be a key driver of tumour progression by inhibiting apoptosis of malignant cells in breast carcinoma . Fibroblast activation protein (FAP), a serine protease that is selectively expressed in reactive fibroblasts of epithelial cancers has also been noted to have a pathogenic role in malignancy . FAP blockade in mice models of pancreatic ductal carcinoma has been shown to induce hypoxic necrosis of tumour and stromal cells in a process that is dependent on Interferon gamma (IFNγ) and TNF alpha (TNFα) . In addition to these effects, reactive stroma and cancer associated fibroblasts are also known to facilitate tumourigenesis and metastasis via generation of oncogenic signals and promotion of angiogenesis .
Acute inflammation
During episodes of acute inflammation, vascular endothelial cells (ECs) are responsible for the immediate recruitment of specific leucocyte subsets to an area of acute pathology by expressing specific adhesion molecules and chemokines in response to biochemical signals generated by inflammatory/infectious/traumatic tissue injury . In the acute inflammatory response to infection, tissue resident macrophages may release cytokines such as IL-1 or TNFα. Along with bacterial components, and activated complement fragments, these cytokines stimulate vascular ECs to express adhesion molecules that interact with surface presented chemokines or other mediators, that, in turn, activate leucocyte integrins . The types and patterns of adhesion receptors expressed as well as the chemokine gradients are determined by the nature of the stimulus driving the inflammatory response . For example, α M β 2- integrin binds intracellular adhesion molecule 1(ICAM-1) to stabilise adhesion and supports migration of neutrophils to the site of acute inflammation . Capturing peripheral blood leukocytes (PBLs) to an area of chronic inflammation in contrast, requires α 4 β 1- integrin to bind to vascular cell adhesion molecule 1 (VCAM-1), a process which is stabilised by the chemokine CXCR3 .
Whilst the EC is the principal cell type controlling leucocyte migration into an area of acute inflammation, stromal components are known to have an important role in the regulation of this response. When co-cultured ECs and fibroblasts are exposed to the pro-inflammatory cytokines TNFα and IFNγ, fibroblasts reduce lymphocyte migration in an IL-6 dependent fashion; these effects are not seen in the absence of TNFα/IFNγ , suggesting that healthy fibroblasts play an immunomodulatory role by preventing inappropriate recruitment of leukocytes to areas of acute inflammation. This is in contrast to the pathogenic role that fibroblasts play in chronic inflammation.
The recruitment postcode
The specific combination of receptors, chemokines and other agents driving leucocyte transmigration is often referred to as the recruitment postcode . When fibroblasts and ECs are co-cultured, alterations in genotype are observed in both cells. These changes are dependent on the anatomical source of the fibroblasts . The phenotype of the fibroblast itself however, is also altered by inflammatory stimuli . It is therefore probable that the fibroblast, rather than the endothelial cell is the primary protagonist in modulating tissue-specific, stimulus-specific inflammatory responses through control of endothelial cell function.
Fibroblast activation
Fibroblast activation refers to the process by which fibroblasts rapidly produce cytokines, chemokines, prostanoids, large amounts of ECM proteins (both constituents and degradation proteins) and express α-smooth-muscle actin in response to tissue injury . Activated fibroblasts are found in damaged, inflamed or healing tissue. These activated cells regulate the behaviour of leukocytes that are recruited to damaged tissue. CD40-CD40 ligand interactions is known to regulate this process by activating the nuclear factor (NF)-κB family of transcription factors, leading to myofibroblast production of large amounts of IL-6, IL-8, cycloxygenase-2 (COX-2) and hyaluronan . Cytokines and chemokines produced by fibroblasts appear be specific to the type of injurious stimulus and the anatomical site of pathology .
The stroma
Tissue stroma comprises the subendothelial basement membrane and the underlying connective tissue containing stromal cells such as pericytes, fibroblasts and organ specific stromal cells (astrocytes, hepatocytes) and epithelial cells as well as adipocytes . The extracellular matrix that lies between stromal cells is comprised mainly of gylcosaminoglycans and proteoglycans. Lymphatic and blood vessels and nervous tissue is interspersed throughout the stroma. It is through this matrix that leucocytes migrate to and from an area of inflammation . Considerable effort has been expended in targeting vascular endothelial cells in cancer and inflammation. While new anti-angiogenic agents have shown promise in some cancers, their application to autoimmune diseases remain disappointing.
Fibroblast biology
The fibroblast is the most common cell type within the stroma . Fibroblasts are identified by their spindle-shape morphology, expression of interstitial collagens (types I and III) and their adherence to plastic in culture . Fibroblasts express numerous markers which aid in their identification ( Table 1 ). However None of these markers is highly specific to fibroblasts, nor is any marker reliably present in all fibroblasts . Because of this, there has been until recently a lack of information on the origin, function and role of fibroblasts in disease .
| Marker | Function | Fibroblast subtype | Expressed elsewhere |
|---|---|---|---|
| Vimentin | Intermediate filament associated protein | Miscellaneous | Endothelial cells, myoepithelial cells, neurons |
| α-SMA | Intermediate filament associated protein | Myofibroblasts | Vascular smooth muscle cells, pericytes, myoepithalial cells |
| Desmin | Intermediate filament associated protein | Skin fibroblasts | Muscle cells, vascular smooth muscle cells |
| FSP1 | Intermediate filament associated protein | Miscellaneous | Invasive carcinoma cells |
| Discoidin-domain receptor 2 | Collagen receptor | Cardiac fibroblasts | Endothelial cells |
| FAP* | Serine protease | Activated fibroblasts | Activated melanocytes |
| α1β1 integrin | Collagen receptor | Miscellaneous | Monocytes, endothelial cells |
| Prolyl 4-hydroxylase | Collagen biosynthesis | Miscellaneous | Endothelial cells, cancer cells, epithelial cells |
| Pro-collagen-1α2 | Collagen-1 biosynthesis | Miscellaneous | Osteoblasts, chondroblasts |
| CD248* | Unknown | Miscellaneous | Pericytes |
| VCAM-1 | Cell adhesion | Miscellaneous | Activated endothelial cells |
| gp38* | Invasiveness in cancer | Miscellaneous | Alveolar cells, podocytes, endothelial cells, neurons |
| Cadherin-11* | Homotypic adhesion | Miscellaneous | Nil |
| Recruitment | TLO formation | Retention | Reduced emigration | Reduced apoptosis |
|---|---|---|---|---|
| IL-1 | gp38 | IFN-1 | CXCL12 (SDF-1) | Bcl-x L |
| IL-6 | CD157 (BP-3) | IL-15 | CCL21 | BCl-2 a |
| TNFα | IL-7 | BAFF | IFN-β | |
| COX-2 | CXCL13 (BLC) | CXCL12 (SDF-1) | NF-κB | |
| CXCL8/IL-8 | CCL19 | CXCR4 | IL-2 | |
| CCL5 | CCL21 (SLC) | TGF-β | IL-4 | |
| CXCL1 | ICAM-1 | IL-15 | ||
| CXCR2 | VCAM-1 | BAFF b | ||
| CXCL5 | APRIL b | |||
| CXCL12 (SDF-1) | IL-6 b | |||
| CXCL12 b (SDF-1) |
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