Modulators in rheumatic disease, and the biologic therapies

Part 1

Immunology - an Update.
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Treatment of the rheumatic diseases has undergone a remarkable change over the last 10 years. This is because of an increased understanding of the immune system and the mechanisms and causes, of rheumatic diseases. We now treat the underlying cause aggressively and early.  With that, we do not see the progressive deterioration in joints that was commonplace 10 years ago. Accordingly, we now require early diagnosis, to ensure early intervention with disease modifying therapy.

Unfortunately, the diagnosis is often delayed. In a study of 174 rheumatoid arthritis patients presenting to our own clinic, the mean time, before diagnosis was 2.01 years, and the diagnosis was made by the referring doctor in only 10% of the cases prior to the referral. This suggests that education is critical, as time wasted before diagnosis represents damage.

The main immune mediated inflammatory joint problems include seropositive and seronegative rheumatoid arthritis, the spondyloarthropathy group including; Ankylosing Spondylitis, psoriatic arthritis, enteropathic arthritis and reactive arthritis.  Of the main immune mediated systemic diseases, lupus, Sjogren’s and the vasculitides, provide targets for the immune treatments now available.

In the immune mediated diseases of bone and joint, inflammation is characterized by pain, swelling, morning stiffness and fatigue.  With damage, the symptoms change and pain tends to occur through the day, with activity, and stiffness is usually only transient and associated with gelling or post rest stiffness. On examination, with active inflammation, the joints are noted to have swelling or heat possible effusions and tenderness.  Early on, there is no joint damage and x-rays will show a few or no erosions.  More sophisticated scanning including magnetic resonance is now available to show early potential erosive disease. Progressive disease however, is associated with increasing thinning of the bone, erosions and joint damage.

The mechanism of inflammatory arthritis: This is associated with a genetic predisposition, and an interaction with environmental factors, that caused the immune system to activate against self in a process called autoimmune disease.

Immune responses occurred by an interaction between a receptor and a Ligand.  The shape of the receptor and the Ligand are critical.  The strength of interaction is called affinity. Receptors are displayed on the cell surface, or may be soluble in the circulation.  The Ligands may be expressed as cell surface molecules such as on the surface of microbes or as soluble molecules.  For examples secreted products of cells.  A grouping of Ligand may be recognized by the immune cells are known as antigens.  The basic recognition unit of the antigen is called the antigenic determinant or epitope.  Different lymphocytes may recognize different epitopes on the same antigen.

Antigens that promote immune response called immunogens.  Antigens that promote an immune response only when combined with another immunogenic carrier molecule, but not on their own, are called haptens.  Antigens that suppress an immune response, and thereby render the immune system to become selectively unresponsive to re-exposure of the same molecule, are called tolerogens.

Genetic factors: There are 23 chromosome pairs in every human cell. Gene mapping has been researched over the last 10 years in what is called the Genome project, and this has enabled characterization of virtually the entire human genome

hla class 1The immune system involves many genes, but the most important gene complex, associated with autoimmunity, is the HLA  histocompatibility complex, located on chromosome 6.  There are three classes of these HLA genes, of which two are involved with the immune system.

Class 1 genes:  these include HLA A,B and C – the so-called classic genes and these are the main actors in the immunological pathways. They are located and expressed in most somatic cells. The level of expression however varies depending on the different tissues. The class 1 molecules identify self versus non self -  and constitute immune markers that enable the body to detect all foreign molecules – called antigen that are foreign to one’s own tissue.  The class one genes are located on most of our own cells.  These genes therefore declare to circulating immune cells, that that particular tissue or cell is part of one’s own tissue.  The immune cell therefore does not react to that tissue.

hla class 2Class 2 genes: these include HLA D genes and are divided into five families – M., O, P., Q. and R . These code for two chains – A and B. they are located in a subgroup of the immune cells that include B -cells, activated T-cells, macrophages, and dendritic cells and thymic epithelial cells.

The function of class one and close to molecules, is the presentation of short pathogen derived peptides to T-cells, a process that initiates the adaptive immune response. Class 2 molecules, are expressed by immune cells and are required to amplify the immune response via intercellular communication.

Innate immunity and the innate immune system.

The immune system has an immediate mechanism to respond to attack.  This mechanism does not require previous exposure and requires no need to adapt to the invading agent.  This system is called innate immunity. It therefore provides the first defence against attack.  It provides the same response to any antigen exposure and lacks immune memory.

It includes the phagocytic cells, inflammatory mediators released by immune cells, chemical mediators and natural killing cells.phogocytic cells

The phagocytic cells include the neutrophils, and monocytes and macrophages.  These cells consume organism or foreign material, and use intracellular chemicals such as superoxide lysozyme’s and nitric oxide to kill and dissolve the foreign matter. They have receptors for antibodies, foreign carbohydrates such as bacterial cell walls and immune agents called complement, which assist in presentation and killing of foreign matter, and antigen. The receptors surface markers which allow binding of the foreign antigen to the cell wall, enabling the cells to process and deal with that antigen. To recognize something different from self, requires pattern recognition receptors.  These are usually a genetically stable set of receptors, evolutionary selected to recognize and bind structures of distant related organisms or produced by the host cells in response to stress or injury.  The structure of these receptors are directly encoded in the genome and transmitted across generations.  Some pattern recognition receptors such as the toll like receptors are found on the membranes of various cell types, while others such as complement molecules exist in soluble form.

 Inflammatory mediators are released by basophils, mast cells and eosinophils. These cells have receptors with high affinity for a class of immunoglobulin called IgE.  They are involved with allergy.

 

Chemical mediators include complement, cytokines which chemicals that communicate between cells, and the adhesion molecules – which traffic or direct neutrophils and phagocytic cells to sites of inflammation.

 

Natural killer cells. These cells have receptors to detect the HLA class 1 molecule or other common or ubiquitous cell surface markers.  By recognizing HLA class 1 on the cell surface, they interpret that cell as part of self and do not react. However if for any reason, the HLA class 1 molecule is not identified on the surface of a cell, that cell is immediately attacked and destroyed by the natural killer cell. If for any reason the HLA class 1 molecule is lost by a cell, such as what happens with malignancy or viral infections, the malignant cell or virally infected cell is depicted as abnormal and destroyed.  The natural killer cell binds to the abnormal cell and injects cytotoxic enzymes into the affected cell and kills it. In addition there are pattern recognition molecules on the walls of pathogens that are recognized by the immune system as ubiquitous molecules foreign to self. Immune cells have pattern recognition receptors to recognize these foreign molecules. These receptors include the Toll – like receptors, TLR’s, that bind to pathogen and induce nuclear signaling for cytokine production. Host cells under attack may down-regulate the class 1 molecule expression on the surface. In addition, host cells can express signal molecules that suggest the cell is stressed and under attack. These include heat shock proteins and also two molecules called the MICA and MICB. These are detected by the Toll like receptors, which activate the killer cells killer activating receptors, KAR’s and result in an attack on the affected cells.

The acute inflammatory response:

In infected tissue for example, activating substances are released by virus, bacteria and damaged tissues.  These activating substances, cause  adhesion molecules, to express on the endothelial wall of circulating blood vessels. Circulating neutrophils bind to the molecules, called Sialyl Lewis molecules, which causes the neutrophil to roll closer to the membrane.  neutrophilAt this point, a second adhesion molecule called E-selectin, on the endothelium, binds to a receptor on the neutrophil called integrin, resulting in adhesion of the neutrophil to the inner endothelial surface. The binding is enhanced by cytokines, especially interleukin 1, and tumour necrosis factor alpha. TNFα. The neutrophil, then squeezes through the endothelial wall and moves to the tissue outside of the blood vessel, in a process called diapedesis. Once in the tissue, the neutrophil is able to devour the foreign invading bacteria or substance, in a process called phagocytosis.  The process of phagocytosis is enhanced by coating of the foreign bacteria with complement.

The Complement cascade. complement cascade

This provides an inmate immune mechanism to enhance binding and killing of foreign invading organisms or antigen. There are two pathways in this mechanism. The classical complement pathway and the alternative pathway.

They both provide a chain reaction, starting with precursors of complement and ultimately the development of attack complexes for killing of foreign invaders.

C3 convertase causes hydrolysis of C3 into C3b and C3a fragments.  See three be finds to the surface of pathogens leading to enhanced phagocytosis – opsonisation. C3b then cleaves C5 into C5a and C5b fragments.  C5a enhances chemotaxis, and both C3a and C5a are anaphylactoid - triggering mast cells and increasing vascular permeability and smooth-muscle contractility. C5b initiates the membrane attack pathway. C5b combines with C6, C7 C8 and C9 and together form the membrane attack complex. The membrane attack complex causes osmotic lysis of the target cell.  The cell swells and is destroyed by bursting.

The classical complement pathway: this is a specific immune response and requires antibody and antigen binding to the C-1 complex. The scene one complex then splits into C2a and C4b fragments, which activate C3 convertase. C3 convertase can then initiate the complement cascade.

The alternative complement pathway: this mechanism is non-specific, and causes direct activation of C3 into C3a and C3b, by hydrolysis of C3, by bacterial cell surface C3b receptor. Hence complement activation can then occur independent of the presence or absence of antibody.

The prostaglandins. Prostaglandins are a series of chemicals that mediate the immune response in several ways.  They are formed from production of arachidonic acid by conversion from phospholipids by phospholipase A2.  This process is stimulated by direct stimulus, enhanced by cytokines – especially IL-1, Tumor necrosis factor and growth factors.  It is reduced by corticosteroids and inhibitory cytokines such as I L4.  Arachidonic acid is converted by cyclooxygenase enzymes to prostaglandin H2, and then further to prostaglandins D2, E2, I2, F2α, I2 and thromboxane A2. Each of these prostaglandins perform different functions in the immune system.

E1, E2, F1α and F2 α are associated with increasing vascular permeability, causing inflammation with weal and flare response to trauma.  They contract smooth muscle in the uterus gastrointestinal tract and the bronchus and increase hyperalgesia in sensory afferent nerve fibres.  They also reduce gastric acidity.

D2 increases hyperalgesia and inhibits platelet adhesion.

Thromboxane increases vascular permeability. It is primarily located on the platelet, and it is the main factor that aggregates platelets, and therefore facilitates clotting.

Prostacyclin PG I, decreases platelet adhesion and decreases vascular tone.  This is located in the endothelium and maintains the integrity of the endothelium and prevents clotting on the surface of the endothelium.

Arachidonic acid is also oxygenated through the lipoxygenase pathway, to form the leukotrienes – B4, C4, D4 and L4. These are also involved in mediation of allergies.

The adaptive immune response –acquired immunity.

lymphocytesThe primary cells that mediate this process are the antigen presenting cells, T-cells and the B cells

The adaptive immune system has the capacity to modify or adapt its response.  Immunological memory allows enhanced responses on repeat encounter of the appropriate antigen.  Both T-cells and B cells are able to modify their receptors or antibodies respectively through genetic transformation.  Rearrangement of DNA or somatic mutation can result in an enormous number of different receptors or antibodies.  Each cell produces only a single type of receptor or antibody, but the total number of cells and pool of receptors becomes exponential.  Each cell generates the receptor in a random manner.  Some cells developed structures capable of receiving recognizing self and removed. Others become selectively formed because of clonal selection.  The immune system therefore becomes more efficient with re-exposure, and with subsequent divisions.

T-cells and B cells form from pluripotential cells in the foetal liver and marrow. B cells matured in the bone marrow whilst T-cells migrate to the thymus. Both develop adaptive responses in secondary lymphoid tissue, including the lymph nodes, the spleen and the mucosal lymphoid tissue. T-cells travel to periarteriolar lymphoid sheaths. The B-cell responses occur in a mesh work of dendritic cells within lymphoid follicles. On contact with antigen, B cells develop responses in germinal centres.t cell receptor

To prevent these B cells and T-cells and immune cells reacting against self, the body has developed a system of tolerance.  Central tolerance involves the negative selection after self antigen recognition. T-cell clones that recognize self are deleted in the thymus.  B -cell clones that recognize self are deleted in the bone marrow.

Peripheral tolerance, involve also clonal deletion of autoreactive B cells. Circulating regulatory CD4 and CD 25 T-cells, called Tregs, patrol and delete autoreactive cells.

T-cells:

The surfaces of T-cells have receptor markers.  These are molecules that constitute receptors that recognize antigen or other cell molecules and initiate cellular processes.  By convention, these cell surface molecules are assigned a number called the cluster of differentiation – CD. .  The presence or absence of these cell surface molecules, determine the function of the cell.  All T-cells carry CD3.

There are two main groups of T-cells.  These are the CD4 and the CD8 T-cells.

 CD4 T-cells: These bind to MHC class 2 molecules. th1 killerThe class 2 molecules are normally expressed by a subgroup of immune cells that include B cells, activated T-cells, macrophages, dendritic cells, and thymic epithelial cells.  These cells produce cytokines that amplify the immune response. CD4 T-cells usually act as helper T-cells and recognize antigen presented by dendritic cells with MHC class 2 molecules.  They produce cytokines.  There are two subtypes of the CD4 T-cell. These are the type 1 – TH1 helper cell and the type 2 – TH2 helper cell. TH1  helper T-cell is secrete interferon-gamma and interleukin-2, which activate macrophages and cytotoxic T-cells to  kill intracellular organisms.  TH2 helper  T-cells secrete interleukin 4, 5 and 6 which help B cells secrete protective antibodies.  The B cells recognize antigen either directly or in the form of immune complexes on follicular dendritic cells in germinal centres.

CD8 T-cells: These bind to the MHC class 1 molecule.  Class 1 genes are expressed by most somatic cells.  Level of expression varies depending on the tissue. CD 8 T-cells are cytotoxic. Class one genes enable the process of self recognition.  Absence of class 1 genes initiates killer cell activity. CD 8 T-cells are usually cytotoxic and recognize antigen presented by MHC class 1 molecules. Some CD 8 T-cells have a suppressor function. 

Antigen presenting cells: these cells include the dendritic cells and macrophages primarily, although lymphocytes including T-cells B cells plasma cells and memory cells have the ability to present antigen. The antigen presenting cell internalizes  the antigen, and internal processing occurs, followed by presentation of extracts of the original antigen on the surface of the antigen presenting cell. This is then presented as a complex, together with HLA class 2 molecules to T-cells. The T-cells then activate.  Co-stimulatory molecules are expressed on the dendritic cell and enhance response by binding to  molecules on the T-cell surface. This increases cellular connectivity and proximity.

For example CD 28 binds to B7.  CD 154 (CD40-Ligand) binds to CD 40.  CD 2 binds to CD 58.

th2 responseT-cell activity: most cytotoxic T-cell are positive for CD 8 and recognize processed antigen presented by MHC class 1 molecules and kill infected cells, thereby preventing viral replication. Activated cytotoxic T-cells secrete interferon-gamma and set up a state of resistance to viral infection.

Cytokine production:

Multiple cytokines have been described.

Interleukin 1.This is produced by macrophages, and results in activation of T-cells and macrophages with promotion of inflammation. They are implicated in inflammatory arthritis.

Interleukin-2: this is produced by type 1 help T-cells – Th1 lymphocytes.  It is involved in activation of lymphocytes and natural killer cells and macrophages.

Interleukin 4:  This is produced by type 2 helper T-cells – Th2 cells.  Mast cells, basophils and eosinophils and activates lymphocytes and monocytes.

Interleukin 5: from type 2, Th2 helper T-cells mast cells and eosinophils.  Is involved in differentiation of eosinophils and allergy.

Interleukin-6 produced by type 2 Th2 helper T-cells and macrophages and is involved in activation of lymphocytes and differentiation of B cells and antibodies.  Is a strong stimulates of the acute phase proteins in the liver such as C-reactive protein.  This is strongly involved in inflammatory arthritis and erosive disease.

Interleukin 8: produced by T-- cells and macrophages and is involved with chemotaxis of neutrophils, basophils and T-cells.

Interleukin 11: produced by bone marrow and stromal cells and stimulates accuse phase protein and interferon by Th1 helper cells and natural killer cells.  Inducers type 1, Th1 helper T-cells.

Tumour necrosis factor β:  produced by macrophages, natural killer cells, T- cells, B cells and mast cells.  This is a major promoter of inflammation.  Strongly associated with rheumatoid arthritis.

Lymphotoxin – tumour necrosis factor β: produced by the type 1 helper T-cells and B cells.  Strong promoter of inflammation.

Granulocyte macrophage colony stimulating factor: produced by T-cells, macrophages, killer cells and B cells.  Stimulates production of leucocytes and granulocytes and monocytes.

Transforming growth factor β.  Produced by T-cells, macrophages, B cells and mast cells.  Is involved with immunosuppression.

Interferon g: produced by the type 1 -- Th1 helper T-cells and natural killer cells.  Activates macrophages, inhibits type 2 - Th2 helper T-cells.

Interferon β: Produced by virally infected cells and induces resistance of cells to viral infection.

Interferon α: Produced by virally infected cells and induces resistance of cells to viral infection

B-cell function.

B cells arise from pluripotential stem cells in bone marrow.  b cell receptorThey arise from two distinct lineages.  B1 cells dominate in the pleural and peritoneal cavity and B2 cells are distributed throughout the lymphoid organs and tissues. Each B-cell is specific and produces immunoglobulin of only one antibody recognizing only one epitope. Immunoglobulin molecules found within, and are displayed on the cell surface, functioning as B-cell receptors.

The B cell receptor has heavy and light chain molecules.  Each has a variable-N-terminal end, at the epitope binding site.

 This variable region generates the overall diversity of the immunoglobulin response. Random division of cells, results in a massive diversity amongst the B cells.

B cells are able to terminally differentiate into plasma cells.  The plasma cells are immunoglobulin producing and secreting cells.  They produce large quantities of immunoglobulin during a relatively short lifespan of approximately 1 month. The circulating immunoglobulins constitute the antibodies of the immune response.

B cells can be activated by antigen presenting cells, TH2 T-cells, and antigen directly.  Activated B cells divide and mature into short lived, plasma cells secreting IgM, or into long-lived plasma cells, which secrete a range of immunoglobulin, or develop into memory cells.  With increased divisions of these cells there is affinity maturation of the B-cell receptors and antibodies. Antibody or receptor diversity rises by division of the variable region in the N-terminal chains of the immunoglobulin. There are five different classes of antibodies.  These are IgA, IgD, IgE, IgG, and IgM. The class of the antibody is defined by the C-terminal region -- the constant region of the immunoglobulin molecule.b cell activation

B cells have different surface markers for different stages of development.  Stem cells develop into pro-B cells, which carry CD10 and CD 38, and as they develop into pre-B cells, express CD 19 and CD 24 and CD 20 on the surface.  Immature B cells will manifest CD 19, CD 20 and CD 24 and CD 39.  Activated B cells express CD 10, CD 19, CD 20 and CD 39.  Memory cells express CD 19, CD 20 and CD 39.  Plasma cells express CD 38.  Because of the varying surface markers, B cells can be targeted by therapy to affect only certain stages of development.  For example, Rituximab will target CD 20 cells, but will leave other B cells unaffected.

 Activated B cells produce cytokines, TNF α; interleukin-6 and Lymphotoxin α. Lymphotoxin promotes the formation of new lymphoid structure in the synovium.  The cytokines promote immune amplification.

 B and T-cell receptor diversity

Heavy chain of B cell antibody and receptor are located on chromosome 14.  Light chain Kappa is located on chromosome 2 and light chain lambda on chromosome 22.

T-cell receptor for A and D are located on chromosome 14, for B on chromosome 7 and for G. on chromosome 7

The genes include V., D., J. and C. regions. V genes code for the variable region. D for the diversity region, .J. for the joining region and C for the constant regions

Each lymphocyte uses a different gene combination to form the genetic code of the receptor.  These gene rearrangement induced by recombination activation genes – RAG genes. The recombination process is subject to splicing and insertions of additional nucleotides around the V, D, and J. junctions before they are ligated. Splicing errors and added nucleotides further increase diversity.  As a consequence there is an almost infinite potential for creation of molecularly unique receptors.  A series of nucleases and ligases, do the cutting and pasting of the different gene segments. plasma cell

antibodySimilarly, antibodies obtained their diversity by clonal selection processing amongst V,D,and J. regions. The gene arrangements are induced by recombination activating genes. And again, recombination process is subject to splicing and insertions to increase diversity, and impart a molecularly unique receptor. After antigen contact with co-stimulatory signals, the B-cell differentiates into plasma cells which secrete antibodies. The stronger the antigen binding, the greater the chance of B cells survival to produce high affinity antibodies. B-cell proliferation will occur in germinal centres of lymphoid tissue.

Autoimmunity.

Autoimmunity is a clinical syndrome caused by the activation of T-cells or B cells or both, in the absence of an ongoing infection or other discernible cause.  There is a breakdown of tolerance with defective T-regulation function, and enhanced activation of T-cells and B cells.  There is a genetic component.  There are multiple susceptibility genes in which the HLA gene system is the commonest factor. In the spondyloarthropathy, the B27 gene increases susceptibility.  In rheumatoid arthritis HLA-DR4 for increases the risk of disease by a multiple of 4. HLA DRB1*0401 and DRB1*0404 alleles are strongly associated with a more aggressive rheumatoid arthritis.

In addition to the genetic component, there is an environmental component. This may include potential infectious agents, molecular mimicry or tissue damage and self antigen exposure.  The defective clearance of apoptotic cells may also result in persistence of antigen, which is able to stimulate the immune system against self.

Thereafter, the immune system may either be organ specific or organ non-specific.

Biologic interventions.

With the knowledge of the immune system, immune therapies are now designed to block or even enhance certain components.  Therapeutics with anti-TNF (Adalimumab, Humira and infliximab, Remicade) or cytokine blockers (IL-6 – Tocilizumab, Roactemra), or blockers of co-stimulatory molecules (Abatacept, Orencia), are already therapeutically available and have shown dramatic response in autoimmune disease. Anti-B-cell antibodies such as Rituximab have been used in treatment of rheumatoid arthritis. In addition, soluble receptor s, such as soluble Tumor necrosis factor receptor, which binds excessive circulating TNF, may be simulated by therapeutic blockers (etanercept, Enbrel), and soluble IL-1 receptor antagonist, that binds to IL-1 receptor and prevents the exposure to the receptor of over abundance of IL-1has been manufactured therapeutically as Anakinra, Kineret.

In this way therefore, fundamental treatment of the immune aberration of autoimmune disease is being developed and promises hope for millions of sufferers of autoimmune disease worldwide in the future.

Go to part 2 of this article - Biological modulators of the arthritis diseases - Biologic therapy

Dr D. Gotlieb.
drdoc on-line
January 2011.

Revised Aug 2011

Copyright protected.

 
   
   

Dr David Gotlieb
Original Article : copyright
Cape Town
South Africa
August 20
11


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