Assignment #2 - Research Article Summary and Critique

Type IX collagen deficiency enhances the binding of cartilage-specific antibodies and arthritis severity

Stefan Carlsen, Kutty Selva Nandakumar and Rikard Holmdahl


Summary

Rheumatoid arthritis (RA) affects joint articular cartilage, synovium and bone by joint-specific autoimmune attack. This inflammatory disease is degenerative, leading to loss of function and joint deformity. In this article, researchers use animal arthritis models to determine whether the severity of correlated rheumatoid arthritis is dependent on the preexisting cartilage quality.

As mentioned in the previous blog, articular cartilage (in synovial joints) is of the hyaline cartilage type. Hyaline cartilage’s strong yet mildly flexible physical characteristics can be attributed to the type II collagen fibers that are most predominant in hyaline cartilage. Type IX and type XI collagen fibers are also found in hyaline cartilage, and it is thought that type IX collagen provides the necessary association between collagen fibrils and matrix macromolecules for integrity and stability of the cartilage. Type IX collagen associates with type II collagen by forming three polypeptide chains that periodically cover the surface of the collagen II/collagen XI heterofibril. (Carlsen et al.) Therefore, by disrupting or arresting the formation of one of the collagen IX polypeptide chains (α1) in a group of transgenic mice, the resulting absence of collagen IX can be used to test the contribution of collagen XI to the long-term stability of cartilage, as well as the vulnerability of the defective cartilage to inflammatory attack by monoclonal antibodies.

In order to simulate the susceptibility of collagen XI deficient mice to the rheumatoid arthritis inflammatory disease, collagen XI development was prevented in mice by backcrossing a transgenic disruption of the col9a1 gene. In addition, researchers used three animal models for arthritis; collagen-induced arthritis (CIA), collagen-antibody-induced arthritis (CAIA) and stress induced arthritis (SIA).

The CIA disease model has a similar disease course to that of RA, and therefore was used to test whether or not preexisting cartilage disorder changed the course of rheumatoid arthritis by allowing better accessibility to the immune response macrophages and neutrophils. CIA was induced in mice by injection of type II collagen from rat chondrosarcoma. The severity of the resulting arthritis was assessed by observation of the four paws, and assigning numbers to swelling or redness observed in one toe, more than one toe/ankle affected, or the whole paw affected. The two assay collagen IX deficient mice of different genetic backgrounds were found to develop more severe arthritis than of the control mice. In addition, they measured no change in antibody titers (a measurement of how much antibody was produced that recognizes a particular epitope) reinforcing that no new T-cells were produced to prime the inflammation because CIA disease is T-cell independent.

The CAIA model, inflammation is mediated primarily by neutrophils and macrophages, and therefore researchers could use this model to study the inflammation while ignoring the dependence of the immune response on initial complementary binding of Fcγ receptors. In addition, the regulatory role of T and B cells in cartilage stability can be investigated because it is known that with the absence of either T or B cells, the CAIA disease is enhanced. CAIA was induced by injecting the mice with purified IgG from B-cell CIIC1 antibodies (which binds to the C1 epitope [antibody recognition region of macromolecule]). Arthritis was observed to develop rapidly in both collagen IX deficient mouse strains, even affecting the knee. After further extending the study to test for the effect of different types of antibodies on arthritis, it was found that the different antibodies recognized different portions of the C1 epitope, and that antibody response overpowers the immune response (by macrophages and neutrophils) in the CIA model.

The last animal disease model, SIA appears to cause similar symptoms as CIA (edema and deformity of the hind paws) but is distinct because it is T-cell independent and causes proliferation of fibroblasts and periarticular enthesophyte formation in cartilage and bone, which can eventually lead to ankylosis (a joint inflammation). Since SIA is spontaneous and tend to occur in old males grouped together from different litters, SIA was induced in that exact way—grouping male mice together isolated from different litters. As in the above models, the developing arthritis was seen to be more severe in type IX collagen deficient mice than in the control mice.

These findings accentuate type IX collagen’s role in maintaining cartilage stability and integrity, and that genetic disorders which may disrupt cartilage integrity may be more vulnerable to inflammatory attack by pathogenic antibodies due to the increased accessibility of type II collagen fibrils.



Critique


This research paper was very clear to explain the methods and techniques used to draw their conclusions. Though the experiment was quite interesting, it was sometimes difficult to follow due to abundance of biochemical acronyms used.

The conclusions drawn from their experiment strongly supported their hypothesis, and they even chose at times to further extend their research (as in CAIA, by assessing the difference in severity of arthritis as a result of different antibodies) or reinforce their conclusions by retesting on a larger number of mice. In addition, they explored the
direct effect of CIIC1 antibodies on collagen II, by adding CIIC1 antibodies to chondrocytes cultured in vitro. The results observed (collagen II fibril disorganization and increased matrix synthesis) further strengthened their conclusion that the CIIC1 antibodies contribute directly to cartilage damage, and that type IX collagen deficient mice have type II collagen that is more accessible to the CIIC1 antibodies.

An obvious flaw to mention, concerning the processed data, is that the author did not refer to either the tables or figures in the discussion section. Furthermore, it was surprising that the author did not choose to include any photographs of the inflamed mouse paws, any figures depicting histological cartilage destruction, nor any cartoon images to illustrate the structures and interactions of type II and type IX collagen. Overall, this research paper was well organized, and the result tables that were provided were intuitively obvious to understand.

Assignment #1 - My Favourite Tissue - Cartilage

Introduction

Cartilage is a specialized form of supporting connective tissue derived from embryonic mesenchyme (Junqueira, and Carneiro, 2005). It is composed mainly of an extracellular matrix enriched with glycoaminoglycans and proteoglycans (ground substances), and collagen and elastic fibers. By varying these components, three cartilage types (Hyaline, Elastic and Fibrocartilage) can be produced, each of which have different structural properties related to their function and location in the body (Martini, 2006).


1. Hyaline cartilage

(haylos, glass) - appearance: blueish-white and transluscent.

Hyaline cartilage is the most common type of cartilage in the body (as well as the most studied!). It is found as costal cartilage between the tips of ribs and the bone of the sternum. In synovial joints, it covers the bone surfaces (articular cartilage), and the conduction portion of the respiratory system it supports the larynx, the trachea and the bronchi. Hyaline cartilage also forms part of the nasal septum. The primary fiber type found in hyaline cartilage is type II collagen.

Functions:

• Provides strong but somewhat flexible support
• Shock absorber in joints
• Reduces friction between body surfaces in jointsServes as a temporary skeleton in embryos (Junqueira and Carneiro, 2005)
  

All hyaline cartilage (except articular cartilage of joints) is covered by perichondrium. The perichondrium can be divided into two distinct layers;

the outer fibrous layer of dense irregular connective tissue, important for support, protection and attachment of the cartilage to other structures, and

an inner cellular layer important to the growth and maintenance of the cartilage. (Martini, 2006)

2. Elastic Cartilage

appearance - yellowish (due to elastin of elastic fibers)

Elastic cartilage shares many of the same features as hyaline cartilage. Like hyaline cartilage, it contains type II collagen, but elastic cartilage is composed of a larger proportion of elastic fibers that contribute to its extreme resiliency and flexibility. It forms the pinna of the outer ear, the epiglottis, the auditory canal and the cuneiform cartilages of the larynx (Martini, 2006).

Functions:

• provides support, but it is able to withstand damage during distortion, as well as return to its original shape
• Elastic cartilage is often found to be gradually continuous with hyaline cartilage, and it too possesses a perichondrium (Junqueira and Carneiro, 2006).

3. Fibrocartilage

Fibrocartilage is the most durable and tough of the three types of collagen. Relative to hyaline and elastic cartilage, fibrocartilage has little ground substance, and is largely abundant with densely interwoven collagen type I bundles (Martini, 2006). Fibrocartilage contains such a large proportion of collagen that it is considered an intermediate tissue between dense connective tissue and hyaline cartilage. It composes the fibrocartilaginous pads that lie between the vertebrae of the spinal chord and the pubic bones of the pelvis, as well as tendons and within/around joints. (Martini, 2006).

Functions:

• Resists compression
• Prevents contact between adjacent bones that could be damaging.
• Limits relative movement (Martini, 2006)

Taking a Look at the Cellular Level of Cartilage

Cartilage cells are called chondrocytes. They occupy spaces within the extracellular matrix known as lacunae (Martini, 2006). In hyaline cartilage, young elliptically shaped chondrocytes are located at the perifery. Within the matrix, chondrocytes appear rounder and divide mitotically to form clusters of isogenous groups.

As mentioned earlier, cartilage is not vascularized, therefore chrondrocytes located deep within the matrix depend on the diffusion of nutrients from the perichondrium. Since cartilage is flexible mostly located in areas of compression, the pumping action of compression and depression by adjacent structures (such as bones) allow more efficient diffusion of water and nutrients (Junqueira and Carneiro, 2005).




Histogenesis


All three types of cartilage is derived from messenchyme tissue (A). Messenchymal cells begin by retracting their extensions, then they proliferate mitotically to give rise to a highly cellular tissue (B). The cells formed by this process are called chondroblasts, and their abundance in ribosomes allow them to synthesize and secrete the cartilage matrix (C). As this occurs, the chondroblasts become increasingly seperated in the matrix (Junqueira and Carneiro, 2005).

Since cartilage development occurs from the center outwards, the messenchymal cells differentiate to produce cells of the center of the cartilage more characteristic of chondrocytes, whereas those of the perifery are typical of chondroblasts (Junqueira and Carneiro, 2005).

Cartilage Growth

Cartilage growth occurs by either interstitial growth or by appositional growth:

• In interstitial growth, cartilage is enlarged from within by the secretions from daughter chondrocyte cells produced via mitotic division of preexisting chrondrocytes. This is the most predominant type of growth during development and this process beings early in embryonic development, as well as during adolescence (Martini, 2006). This type of growth occurs in the epiphyseal plate of long bones, ultimately to lengthen the bone by acting as a cartilage model to be ossified during endochondral bone formation. Interstitial growth also occurs within articular cartilage to replace the cartilage that may gradually wear away within the moving joint. (Junqueira and Carneiro, 2005).
  
• In appositional growth, perichondrial cells of the perichondrium inner cellular layer undergo repeated cycles of division, and the innermost layers differentiat into immature chondrocytes. These condrocytes secrete the matrix which eventually surrounds them, and within the lacunae they differentiate into mature chondrocytes (Martini, 2006). In appositional growth, the new cartilage is added to the surface of existing cartilage, therefor this type of growth results in an increase of the girth of the cartilage. Though neither interstitial growth and appositional growth occur in normal adults, appositional growth may arise in circumstances of cartilage repair. (Junqueira and Carneiro, 2005).
  
  

Cartilage Pathologies

CHONDROSARCOMA

Chondrosarcoma, the second most common primary type of bone cancer, is a malignant cancer whose tumor cells produce a pure hyaline cartilage that results in abnormal bone and/or cartilage growth (Randall et al, 2003). The malignant cartilage cells usually arise to produce central chondrosarcoma, the where the tumor growth occurs within the center of existing normal bone, beginning in the metaphysis and extending to the diaphysis. The second general type of chondrosarcoma (peripheral chondrosarcoma) is rare. In peripheral chondrosarcoma malignance cartilage cells grow within the cartilaginous cap of a pre-existing osteochondroma (a condition where growth plate forms an outgrowth on the surface of the bone).

Note: Chondrosarcoma describes a type of malignant tumor, which has the potential to be life threatening due to its capability to spread cancerous cells to other parts of the body. Chondroma (also referred to as osteochondroma) is a form of benign tumor. This type of tumor can be easily removed in surgery, and does not have the tendency to proliferate in other tissues of the body (Randall et al, 2003).

The term chondrosarcoma is used to describe a large variety of cartilaginous tumors. The major subtypes of chondrosarcoma can be differentiated based on histological observation (appearance in microscopy) and on the location of the chondrosarcoma in the body.

These CT photographs depict chondrosarcoma affecting the skull of a 64-year old female. Note the large protrusion in the frontal area.

Chondrosarcomas variations:

Periosteal chondrosarcomas
(Rare: 1–2% of all chondrosarcomas)

This type of tumor originates at the surface of the bone (usually the metaphysis of the distal femur or proximal humerus) and develops in the soft tissues as a lobulated mass. It is causes long term tenderness, though the pain is relatively mild. Histological analysis is sufficient for diagnosis of periosteal chondroma. Individuals affected by periosteal chondrosarcomas are relatively young. (Ollivier, 2003)


Mesenchymal chondrosarcomas
(2–3% of all chondrosarcomas)

This form of lesion is quite aggressive, and appears as an undifferentiated cell mass with some well differentiated cartilaginous areas. The most common sites affected are the femur, pelvic bones, ribs and vertebrae. Treatment for this type of chondrosarcoma is relatively invasive; including multidrug chemotherapy combined with surgery and radiotherapy. Despite rigorous treatment, the 10-year survival rate is only 28%. (Ollivier, 2003)

Clear cell chondrosarcoma
(rare form 2%)

This form of low grade lesion is characterized by its cellular identification (cytology), typical localization to the epiphyseal region of long bones with extensions into the metaphysis, and slow evolution. Symptoms include pain and swelling, which may persist for extensive period of time (1 to 23 years). This form of chondrosarcoma appears to predominantly affect males of age 30 to 50, in skeletal regions of the femur, humerus and tibia.

Radical surgery is typically used to combat clear cell chondrosarcoma, and the average 5 year survival rate post-surgery is about 92%.



Dedifferentiated chondrosarcoma
(10–12% of all chondrosarcomas)

Dedifferentiated chondrosarcoma is a more aggressive form of chondrosarcoma, characterized by a special histology and very poor survival rates. Affected individuals usually experience pain and swelling, as well as bone fractures as a consequence to the weakened cartilaginous regions. Dedifferentiated chondrosarcoma typically affects the femur, the acetabular regions and the proximal humerus. Aggressive treatment involves surgery followed by chemotherapy or radiotherapy. As previously mentioned, survival rates are quite poor, with an overall 5-year survival rate of only 8.5–13%.

This photo depicts the existance of two typer of chondrosarcomas at a single location, typical of dedifferentiated chondrosarcoma. In the top image, the cartilage on the left is affected by conventional chondrosarcoma. The asterisk identifies the artifact that seperates the cartilage on the right, which is affented by dedifferentiated components.

Histological observation of the dedifferentiated chondrosarcomas reveals the atypical spindal shaped cells (TEM on bottom left), or osteosarcoma with lace-like osteoid formation (TEM on bottom right).

Secondary chondrosarcoma
(12% of all chondrosarcomas)

Secondary chondrosarcoma is a special form of tumor that develops in pre-existing lesions. This rare form of chondrosarcoma is quite difficult to recognize and diagnose (Ahmed et al.). Most often, the legion from which secondary chondrosarcoma develops is of the form of solitary osteochondroma, osteochondromatosis, enchondromatosis (Ollier’s disease), fibrous dysplasia, Paget’s disease, irradiated bone or synovial chondromatosis (Ollivier et al).

The detection of secondary chondrosarcoma is possible by characteristics such as an increase in size of a pre-existing enchondroma, the appearance of a lytic area with cortical destruction, associated with pain or fracture. In addition, the appearance of cartilage cap thickening and calcification of the surrounding soft tissues via CT or MRI all suggest possible transformation of a pre-existing tumor to secondary chondrosarcomas (Ollivier et al). Like all other types of chondrosarcoma, this pathology seems to be predominant in males.

References:


[1] Ahmed, AR et al. (2003). Secondary chondrosarcoma in osteochondroma: report of 107 patients. Clin Orthop Relat Res. 2003 June;(411):193-206.

[2] Junqueira, Luiz Carlos and Carneiro, Jose (2005). Basic Histology: text & atlas, 11th Edition. McGraw-Hill: New Yourk, USA.

[3] Martini, Frederic H. (2006) Fundamentals of Anatomy & Physiology, 7th Edition. Pearson Education Inc: San Francisco, CA, USA.

[4] Randall, R. Lor and Hunt, Kenneth J. (2003) Chondrosarcoma of Bone. The Liddy Shriver Sarcoma Initiative; Utah, USA