The pathogenesis of rheumatoid arthritis in radiological studies. Part I: Formation of inflammatory infiltrates within the synovial membrane

Abstract

Rheumatoid arthritis is a chronic inflammatory disease with a multifactorial etiology and varied course, which in the majority of patients leads to partial disability or to permanent handicap. Its characteristic trait is a persistent inflammation of the synovial membrane and the formation of an invasive synovial tissue, called the pannus, which in time leads to destruction of the cartilage, subchondral bone tissue, and the soft tissue of the affected joint(s). The pathogenesis of rheumatoid arthritis is complex and involves cells of both innate and adaptive immunity, a network of various cytokines and an immunoregulatory dysfunction. An important role in the discovery of rheumatoid arthritis pathogenesis was played by magnetic resonance imaging, which showed the disease process to extend beyond the synovium into the bone marrow. Many studies have shown a strict correlation between the vascularity of the synovium (assessed through the power Doppler ultrasound and magnetic resonance examinations), bone marrow edema and the clinical, laboratory and histopathological parameters of rheumatoid arthritis. From the current understanding of rheumatoid arthritis, bone erosions could occur from two directions: from the joint cavity and from the bone marrow. With power Doppler ultrasound, as well as in magnetic resonance imaging, it is possible to visualize the well-vascularized pannus and its destructive effects on joint structures and ligaments. In addition, the magnetic resonance study shows inflammatory and destructive changes within the bone marrow (bone marrow edema, inflammatory cysts, and erosions). Bone marrow edema occurs in 68–75% of patients with early rheumatoid arthritis and is considered to be a predictor of rapid disease progression.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a multifactorial etiology and varied course, which in the majority of patients leads to partial disability or to permanent handicap⁽¹⁾. Its characteristic trait is a persistent inflammation of the synovial membrane and the formation of an invasive synovial tissue, called the pannus, which with variable speed leads to destruction of the cartilage, subchondral bone, and the soft tissue of joint(s)⁽¹⁾. The triggers for the disease to enter a destructive phase are not well known, but newest research points to the involvement of environmental factors, dangersignaling molecules, new cytokines, epigenetic gene regulation, autoreactive T and B lymphocytes, dysfunctional self-regulation mechanisms and the aging of the immune system⁽²⁾. Experimental data and clinical observations reveal that the bone marrow also takes part in the development of RA, being the site where activated lymphocytes accumulate and proinflammatory cytokines are released⁽³⁾.

Pathogenesis of rheumatoid arthritis

RA has a complex background that involves cells of the innate and acquired immune systems, immunoregulatory dysfunction and cytokines.

Cells of the innate immunity take part in a quicklydeveloping, antigen nonspecific inflammatory reaction, that prepares the groundwork for the initiation of adaptive immune response and its amplification⁽⁴⁾. This group of immune cells includes leukocytes (neutrophils, precursors of mast cells, monocytes and dendritic cells) as well as cells outside of the immune system, for example fibroblast-like synoviocytes (FLS) and endothelial cells.

Leukocytes, among which neutrophils dominate, are formed in the bone marrow. In RA, even when it is asymptomatic, their production is increased and their half-life in the blood is extended⁽²⁾. Under the influence of cytokines produced by monocytes/ macrophages, fibroblast-like synoviocytes (FLS) and neutrophils, leukocytes migrate into the joint, where they release chemokines attracting Th17 lymphocytes. Moreover, they take part in the inflammatory reaction by producing cytokines, proteases, reactive oxygen metabolites, complement components, etc. It is thought that one of the reasons for the excessive, local leukocyte activity is a catecholamine deficiency, caused by a loss of sympathetic nerves in the synovium⁽²⁾. Mast cells (MC) accumulate in the synovium and after being activated (for example by immune complexes of the citrullinated peptide and its specific antibody, anti-citrullinated peptide antibody, ACPA – see below) they can release many inflammatory mediators (including histamine, tryptase, which protects neighboring cells from apoptosis), and are a rich source of IL-17^(4–7). Monocytes also gather in the synovium, being activated by various proinflammatory factors and differentiating in situ into macrophage-like synoviocytes – MfLS. Dendritic cells (DCs) are produced in the bone marrow, from which they migrate into the blood stream in an immature form, to reach the majority of tissues, where they recognize pathogenic microorganisms and secrete cytokines activating cells of innate immunity. At last they reach peripheral lymphatic organs, where in their mature form they activate T and B lymphocytes by presenting them antigens. DCs activate these cells and in this way, mature DCs begin and direct the adaptive immune response. In RA patients, DCs aggregate in the synovium, in which an ectopic lymphoid tissue forms.

Cells of the adaptive immune system are various subpopulations of lymphocytes. In pathological states, including RA, activated T and B cells are directed at self-antigens, revealing an underlying autoimmunization process⁽⁴⁾. The autoimmunization response is initiated in asymptomatic RA, as autoantibodies are present even 10 years before the appearance of clinical symptoms⁽⁸⁾. A universally-known autoantibody is the rheumatoid factor (RF), presumably formed as a result of polyclonal activation of B-cells, which recognizes the Fc fragment of human IgG. The newest studies emphasize the role of the autoimmunization response elicited by citrullinated peptides, derived from various self-proteins, including cytoskeleton components (filaggrin, vimentin), enzymes (α-enolase), and cartilage elements (aggrecan, type II collagen)⁽⁹⁾. Citrullination involves the enzymatic transformation of the amino acid arginine into citrulline. The most likely factors responsible for increased citrullination of proteins in RA are environmental factors, such as cigarette smoking and an infection with Porphyromonas gingivalis (P. gingivalis), the bacteria responsible for gingivitis. This is the only bacteria that possesses peptidylarginine deiminase (PAD), the enzyme breaking down arginine into citrulline. Moreover, P. gingivalis also infects the vascular endothelium and may be spread via the bloodstream; in vitro this bacteria causes apoptosis of chondrocytes, which supports its involvement in the destruction of cartilage. The citrullination of proteins is increased by smoking, which is the best documented environmental factor not only increasing the developing RA, but also the severity of this disease, correlating with the extra-articular symptoms of this disease and worse response to therapy⁽⁹⁾.

The citrullination of proteins is found in many inflammatory diseases, but only in RA do the citrullinated peptides, as autoantigens presented to T lymphocytes, induce an autoimmune response – the release of specific autoantibodies (ACPA).

In normal conditions, the adaptive immune response develops in the peripheral lymphatic organs (the lymph nodes and spleen), while in RA it also occurs in the ectopic lymphoid tissue, which forms in the synovium of the joints, tendon sheaths and/or bursae. T lymphocytes infiltrating the synovium have notable proinflammatory activity, releasing various cytokines, including tumor necrosis factor (TNF). The pathogenic role of B lymphocytes relies on their release of autoantibodies, presentation of antigens and their participation in the formation of the ectopic lymphoid tissue. They are also a rich source of cytokines, including proinflammatory TNF. T-cells recognize antigens attached to their own HLA molecules. On the other hand, B-cells recognize unprocessed antigens and themselves act as antigen-presenting cells, with the assistance of helper T-cells (Th-T helper). After recognizing an antigen, B-cells transform into plasma cells and secrete antibodies, while Th cells differentiate into functionally distinct subpopulations⁽⁹⁾.

Cytokines are secreted proteins informing cells of a change in the microenvironment and modulating their function⁽⁴⁾. The classic proinflammatory cytokines are IL-1β, IL-6 and TNF. Other cytokines also have proinflammatory activity: IL-15, IL-17 and IL-23. In RA patients, there is an increased concentration of these cytokines in the serum, the joint fluid and the synovium, and this finding correlates with disease activity. The mechanism by which the majority of currently available biological drugs (such as anakinra, tocilizumab, etanercept, infliximab, adalimubab) act, is by neutralizing the activity of these cytokines.

The structure of the synovial membrane

Macroscopically, the synovial membrane (the synovium) is a thin layer of connective tissue, covering the internal surface of the joint capsule, the tendon sheaths and bursas. Its main function is to nourish the joint cartilage by producing joint fluid rich in hyaluronic acid⁽¹⁰⁾. It is composed of two layers, with a more superficial intima layer and a subintima layer below it (fig. 1).

Fig. 1.

Loose connective tissue containing small blood vessels (subintima layer) covered by a single layer of synoviocytes (intima layer). Hematoxylin-eosin staining (H&E), ×100

The intima contains a mere 1–3 layers of cells and lacks a basement membrane, which facilitates the flow of joint fluid between the vessels in the subintima and the joint space. It is composed of extracellular matrix proteins and synoviocytes. Mesenchymal FLS dominate number-wise, and are responsible for the architecture of the intima layer, its remodeling, secreting extracellular matrix proteins and the main components of the joint fluid^{(4, 10)}. The less numerous macrophage-like synoviocytes (MfLS) clean the joint from debris and microorganisms. It is known for a long time that both types of synoviocytes are the main sources of factors maintaining the inflammatory and destructive processes in the joint(s). Rheumatoid FLS participate in forming the intimal layers, by producing the extracellular matrix, remodeling it so as to facilitate the migration of other cells (including MfLS). An in vitro recreated intima layer, containing rheumatoid FLS, produced excess proinflammatory cytokines, chemokines, and enzymes degrading the connective tissue⁽⁴⁾.

The subintima is composed of loose connective tissue, with blood and lymph vessels, nerve fibers, and several cell types, including macrophages, fibroblasts, mast cells, and T lymphocytes⁽¹¹⁾.

The development of inflammatory infiltrates in the synovial membrane

Characteristic for RA is the thickening of the synovium, both its intima and subintima layers. The intima is thickened through mild hyperplasia (increased number of cells) from 1–3 layer(s) up to 8–12 layers, caused by proliferation of FLS and their resistance to apoptosis (fig. 2). It is thought that the signaling pathways involved in neoplastic transformation, stimulated as a result of long-term exposure to an activating factor, play a role in this process. The FLS of RA patients were shown to have increased expression of the MDM 4 protein, which inhibits the activity of transcription factor p53 that serves as a tumor suppressor⁽⁴⁾. This finding suggests an impairment in the signaling pathway preventing neoplastic transformation. Aside from this, rheumatoid FLS bear a certain resemblance to metastatic neoplastic cells; when rheumatoid FLS are injected into mice, they migrate throughout and initiate destructive processes in different anatomic locations⁽⁴⁾. The cause for the thickening of the subintima is the migration and retention of infiltrated cells (MfLS, lymphocytes, mast cells, etc.).

Fig. 2.

The synovial membrane in rheumatoid arthritis. A. Hyperplasia of the intima layer. Several layers of synoviocytes (on the right) and adherent to it inflammatory infiltrates of lymphocytes and plasma cells. H&E staining, ×400. B. Ectopic lymphatic tissue in the synovium. In the vicinity of the hyperplastic intima layers (on the left) abundant inflammatory infiltrates of lymphocytes are visible, with formation of secondary lymphatic follicles (containing the germinal center). H&E staining, ×400. C. Angiogenesis. Fairly abundant inflammatory infiltrates in the synovium, containing lymphocytes, macrophages and plasma cells. Small foci of fibrous necrosis are visible on the surface, as well as growth of tiny vessels (angiogenesis). H&E staining, ×100

Initially it is possible to suppress the inflammatory reaction, if aggressive treatment is introduced early on. Otherwise the disease could take on a chronic, aggressive form, with joint destruction. The critical point, at which the inflammation transforms into a chronic phase, is the activation of endothelial cells with simultaneously increased expression of adhesive molecules and the release of chemotactic factors by synoviocytes⁽²⁾. All of these events initiate a massive infiltration of the subintima by leukocytes, which leak out of the blood vessel endothelium and form infiltrates of different degrees of organization. The extravasation of leukocytes is regulated in part by chemokines. Proinflammatory chemokines attract neutrophils, lymphocytes, monocytes and natural killer cells into the joint. Homeostatic chemokines take part in forming the ectopic lymphoid tissue in the synovium, also attracting B-cells, maintaining the inflammatory response and the destructive processes⁽⁴⁾. The cells infiltrating the joint are persistently activated by various soluble mediators and reciprocal intercellular signaling through surface molecules. The massive accumulation of cells results in edema of the synovium (fig. 3). This way, the cellular infiltrate forms an ectopic lymphoid tissue, which is the site for the local autoimmune response. In around 50% of patients, the infiltrates have a diffuse arrangement, containing T and B-cells, macrophages and dendritic cells. In the remaining patients, the infiltrates resemble follicles of the peripheral lymphatic organs, and in some (approximately 25%) they contain structures similar to the follicular germinal centers, which correlates with a severe disease course^{(9, 12, 13)}.

Fig. 3.

The immune-inflammatory response in a rheumatoid joint (described in the text)

As it stands, the increased vascularity of the synovium is the indisputable sign of the invasive synovial tissue (joint pannus); it results from angiogenesis, the formation of new capillaries from already-existing vessels, under the influence of many chemokines, including vascular endothelial growth factor (VEGF). This stage of the disease is well visible in both PD USG and in MRI, particularly the T1-weighted images after contrast administration, which show the presence of vessels/increased signal from a thickened (hyperplastic and edematous) synovium (figs. 4, 5). Despite the increased angiogenesis in the hyperplastic and metabolically-active synovium, the tissue is in a state of hypoxia. Such conditions sustain the infiltration of this tissue by T-cells and macrophages, the production of chemokines and proinflammatory cytokines, in addition to disturbing vasculogenesis (the process of blood vessel formation from precursor cells of the endothelium, released from the bone marrow). The majority of blood vessels in the synovium are structurally immature, lacking pericytes – cells stabilizing the walls of small vessels. Such immature capillaries possess significant permeability, thus furthering the hypoxic state, extravasation of leukocytes and synovial edema⁽⁴⁾. Moreover, the newly-formed vessels often have problems with vasoregulation, lacking the nerves and receptors necessary for the interaction of neuropeptides, like substance P⁽¹⁴⁾. The distribution of new vessels is also aberrant. A normal synovial membrane, especially its superficial layers, is richly vascularized in order to meet the metabolic needs of the avascular cartilage. However, in RA the vessels are redistributed, so that the deeper layers of the synovium are more vascularized. These changes in the microvascular architecture, alongside the dysfunction of vasoregulation, may worsen the hypoxia, increasing anaerobic processes in the chronically inflamed synovium⁽¹⁴⁾. Angiogenesis also impairs the cartilage, adversely affecting its biomechanical properties. Healthy cartilage does not possess vessels, and even release an array of anti-angiogenic factors⁽¹⁴⁾. In RA (as in osteoarthritis) the developing pathologic vessels infiltrate the cartilage from the side of the bone, probably due to an imbalance between pro- and antiangiogenic factors⁽¹⁴⁾. The chronic inflammation leads to the destruction of successive joint structures. This destruction involves not only the synoviocytes of the pannus, but also inflammatory cells infiltrating the bone marrow. There are three processes which play a key role in this destruction: transformation of the synovium into an enlarging, invasive pannus, metabolic changes in the chondrocytes (with an enhanced catabolic processes in the chondrocytes; relative excess of degradative enzymes with a deficiency of their inhibitors); abnormal catabolic remodeling of the bone tissue (dominated by osteoclast resorption)⁽²⁾.

Fig. 4.

The USG examination with a thickened synovium in the midcarpal joint with enhanced vascularity seen in the PDUS

Fig. 5.

The MRI study, coronal scan T1 FS CE: thickening and enhancement of the synovium in the midcarpal joints.

The changes at this stage of the disease, not only the effects of pannus formation, but also those due to pathological processes taking place in the bone marrow, are very well visible on classic radiography. It is possible to see juxta-articular osteoporosis at bones’ epiphyses and metaphyses, being a result of hyperemia and the presence of inflammatory infiltrates in the bone marrow, subchondral inflammatory cysts (containing inflamed granulation tissue), bone erosions (which form either as a result of the penetration of the pannus into the cartilage/subchondrium, or from a cyst within the bone marrow breaking the continuity of the bone cortex), and narrowing of joint space correlating to the loss of cartilage (fig. 6).

Fig. 6.

X-ray examination of both hands: periarticular osteoporosis, inflammatory subchondral cysts and erosions in the scapholunate joint of the right wrist, bilateral narrowing of the joint space in the radiocarpal, midcarpal and carpometacarpal joints as well as joint space narrowing of the 3^rd-4^th PIP joints of the right hand, 2^nd-3^rd PIP joints of the left hand, and the 2^nd MCP joint of the left hand

The vascularized pannus and its destructive effects on the joint and tendons can be seen in the power Doppler ultrasound examination (PD USG) as well as in magnetic resonance imaging (MRI), particularly in T1-weighted images after contrast administration and fat-saturated T2-weighted or short inversion time inversion recovery (STIR) sequences (figs. 7, 8).

Fig. 7.

An effusion/thickening of the synovium plus erosions in the head of the 3^rd metacarpal bone

Fig. 8.

The MRI study, coronal scan T1 FS CE: thickening and enhancement of the synovium in the radiocarpal and midcarpal joints and inflammatory cysts and erosions in the radius and carpal bones

Clumps of the ectopic lymphatic tissue do not only form in the synovium of the joint cavity, the tendon sheaths and bursae, but also in other places affected by RA. For example, such lesions could be seen in the lung tissue, or very often in the subchondrium of the bone marrow^{(9, 15, 16)} (see part III of the article series). The dominant cell line in the bone marrow are activated B-cells, then T-cells and dendritic cells^(16–18). The lymphocytes may be activated by infection, as bacterial DNA is found in the bone marrow of RA patients⁽¹⁹⁾. Changes in the bone marrow may be seen in MRI, in which the lymphoid infiltrates manifest as bone marrow edema. While in the X-ray examination, geodes (subchondral cysts) and periarticular osteoporosis serve as evidence of the infiltrates⁽²⁰⁾. BME is seen in 68–75% of patients in early phase of RA⁽²¹⁾. Changes in the carpal bones and the metacarpophalangeal (MCP) joints are the earliest and most sensitive prognostic factors for the progression of destructive changes⁽²²⁾. Even in the early phase of undifferentiated arthritis, a finding of BME in an MRI study of the hands along with the presence of ACPA antibodies in the serum, predicts a rapid development of RA with 100% accuracy⁽²³⁾.

These observations confirm that the erosion of bone tissue in RA may take place in two ways: from the direction of the bone marrow and from the direction of the joint cavity⁽²⁾. Evidence for the former has been provided by MRI studies, which captured a new element of RA – bone marrow edema (BME) – that increases the probability of erosion formation 6-fold after 6 years of RA⁽²⁰⁾. The amount of the inflamed synovium is a weaker, yet still significant parameter, correlating with the development of erosions in RA⁽²⁴⁾. The CIMESTRA trial has shown, that BME is an independent, and strongest predictor, for the radiological progression of changes in patients with early RA within 1 year⁽²²⁾.

For rheumatologists, the most important indicator of an aggressive course of the disease is still synovitis. The CIMESTRA studies did not confirm this element of RA to be an independent predictor of erosions. Similar to the findings of Mundwiler et al.⁽²⁵⁾, who studied the prognostic value of inflammatory changes in the midfoot of patients with early RA. In the majority of patients (74%), synovitis was found to be the only abnormality. Only in 6% of those studied did erosions appear. These results showed that isolated synovitis is not a predisposing factor for the development of erosions, thus the prognosis of these patients could be better⁽²⁶⁾. It is possible that such patients may not require as aggressive treatment as do those with BME. The answer to this question requires further prospective studies comparing X-ray, MRI, and USG imaging.