Rheumatology 1 - Pathogenesis of Rheumatic Disease
Anil Chopra
1. Describe the role and clinical relevance of HLA molecules in antigen recognition
2. Outline the mechanisms of autoimmunity and how they apply to autoimmune
rheumatic disease
3. Describe the role of cytokines in mediating inflammation and how they are
manipulated in treatment
4. Outline the role of prostaglandins in inflammation and how their blockade is
exploited in therapy
HLA System
The HLA – human leukocyte antigen system is the term used for the MHC (major
histocompatibility complex) in humans. The MHC (or HLA in humans) is a complex
of genes encoding the cell-surface molecules that are required for antigen presentation
to T-cells and for rapid graft injection. It is subdivided into class I, (A,B,C) and class
II (DR):
– Class I: Presentation of intracellular antigens (viral or intracellular bacterial
proteins) to CD8 (cytotoxic) T cells
– Class II: Presentation of extracellular antigens (most bacteria) to CD4 (helper)
T cells
They are encoded by the major histocompatibility complex (MHC) on chromosome
6 and each locus highly polymorphic between individuals. This means that the loci
constituting the MHC are highly polymorphic; that is, many alternative forms of the
gene, or alleles, exist at each locus. This ensures variation between individuals to
ensure survival of the species.
Function of HLA class I antigens
HLA molecules ensure that healthy cells are not destroyed by the immune system by
cytotoxic T-cells in this case. Cytotoxic T-cells are very chauvinistic – they will not
allow any foreign antigens to remain in the body. In infected cells (infected with
bacterial protein for example), the HLA class I molecule presents the intracellular
antigen to the cytotoxic T-cell which therefore recognises there is a change in the cell
and it (the cytotoxic T-cell) is then programmed to kill the infected cell. The cytotoxic
T-cells then release interleukin (in this case IL-2). Interleukins are a group of
cytokines secreted by leukocytes (T-cells) that primarily affect the growth and
differentiation of various haemopoietic and immune-system cells – in the case of IL-2
cause B-cells (a lymphocyte) to mature in the bone marrow. B-cells express
membrane-bound antibodies, and after interacting with the antigen, the B-cell
differentiates into an antibody-secreting plasma cell and a memory cell.
Function of HLA class II antigens
APC antigen-presenting cell: any cell that
can process and present antigenic peptides in
association with class II MHC molecules and
then deliver a co-stimulatory signal necessary
for T-cell activation. Macrophages, dendritic
cells, and B-cells constitute the professional
APCs.
Diagram shows APC presenting an antigen (the bacterial peptide) to the CD4 (helper)
T-cell, which then releases a number of cytokines (interleukins) which activate other
cells of the immune system – notably cytotoxic T-cells which will then destroy the
APC.
Importance of the MHC
This diagram shows the significance of the
MHC. Here you can see that a transplant cell
which is expressing a “foreign” HLA complex
triggers a cytotoxic T-cell to kill it as the host
sees this cell as foreign. This is HLA
mismatch.
The different HLA systems must be matched in order for successful organ
transplantation. At least 2 out of 4 (A, B, C and DR encoding regions) must me
matched for a successful transplant. Living siblings are most likely to be able to offer
organs.
Different variants of the alleles that code for the HLA proteins can increase
susceptibility to disease:
– HLA B27 Seronegative arthritis
– HLA DR4. Rheumatoid arthritis
– HLA DR3. SLE and organ specific autoimmune disease
Cytokines
The following table shows the most important cytokines involved in rheumatology.
These cytokines are released by immune system cells, and they have an effect on
other immune system cells – causing them to proliferate or mature (in bone marrow)
and differentiate. The purpose of the cytokines is to activate other cells to function in
host defence, for example APCs present an antigen to CD4 (helper) T-cells release IL2 which activates CD8 (cytotoxic) T-cells which will then destroy the APC.
Cytokine released
Cell cytokine is
released from
The effect of the released cytokine…
γ-IFN (interferon)
T-cells
Activates macrophages.
IL-1
Macrophages
Activates T-cells, induces fever, and is also a
pro-inflammatory cytokine – it mediates
inflammatory diseases for example,
inflammation of blood vessels.
IL-2
T-cells
Activates both T- and B-cells.
IL-6
T-cells
Activates B-cells.
Macrophages
It is similar to IL-1, but more destructive, i.e.
is more effective in activating T-cells, and so
the T-cell response is more potent and
destructive (i.e. destroys foreign antigens
more quickly than IL-1).
TNF-α
Autoimmune Diseases
There are 2 main types of autoimmune disease, organ specific and non-organ specific:
• ORGAN SPECIFIC
• NON ORGAN SPECIFIC (cellular
– Diabetes
autoantigens)
– Hashimoto’s thyroiditis
– Systemic lupus
– Grave’s disease
erythematosus (SLE)
– Pernicious anaemia
– Rheumatoid arthritis
– Multiple sclerosis
– Sjogren’s syndrome
– Inflammatory bowel
– Polymyositis
disease
– Scleroderma
– Myasthenia gravis
– Vasculitis
There are two types of cellular auto-antigens…
 Organ specific…
– These are things such as cellular enzymes, hormones or receptors that are
specific to the organ affected.
 Non-organ specific…
– These are antigens that are not specific to a particular cell type, for
example (all of the following are common in lupus)…
 Nuclear antigens (DNA and nRNP for example).
 Cytoplasmic antigens (cRNP and enzymes for example).
 Cell-membrane antigens (phospholipids for example).
SLE Systemic lupus erythematosus: photosensitive rash that spares the nasolabial
folds. The autoantigens include DNA itself, histones (part of the DNA structure and
protection), nuclear ribonucleoproteins (involved in splicing and transcription) and
membrane phospholipids.
Genetics of SLE:
• Complement deficiencies (rare) strongly presdispose to SLE
• SLE linked to DR3 (a gene itself linked to C4 deficiency)
• C-reactive protein knockouts associated with lupus
NB: complement and CRP are “hoovers” of the body
Pathogenesis
- Healthy cells (skin) are acted on by either viruses of UV light causing them to
die by apoptosis.
- They do this by displaying nuclear antigens on their cell surface.
- Antibodies, with the aid of complement and C-reactive protein destroy the
cell.
- In SLE if there is failure to clear the apoptotic material, then antinuclear
antibodies can arise.
- These can form immune complexes containing antigen, antibody and
complement.
- These can circulate and deposit in:
o Kidney: glomerulonephritis
o Skin: rashes
o Vessles: vasculitis
Rheumatoid Factors:
- Destruction of joints (Improvement with TNF and IL-1 inhibitors) is caused
by the presence of rheumatoid factors.
- Rheumatoid factors are found in patients with SLE.
- When rheumatoid factors bind to synovial macrophages, they release a
number of cytokines (IL-2, IL-6, IL-1 and TNF).
- This results in high levels of TNF in the joint the induction of
metalloproteinases and eventually leads to rheumatoid arthritis.
- Rheumatoid arthritis improves with TNF inhibitors
Detection:
- autoantibodies are detected by immunofluorescence of cell or organ or by
ELISA
o This is when antibodies are added to sample cells.
o Then add fluorescein conjugated anti-Human IgG
o If there are anti-nuclear
antigens, then the nucleus
fluoresces under UV light.
- This method is generally used to
confirm a prediction rather than
make a diagnosis.
- DNA antidbodes tend to fluctuate
with diease, but all other
autoantibodies stay at a similar level.
-
-
-
Prostaglandins are also found in rheumatic disease
therefore, inhibition can lead to improved
prognosis:
Combined COX-1 and COX-2 inhibitors.
o Eg Aspirin, Ibuprofen, Naproxen,
Diclophenac
o Anti-inflammatory, antipyretic
and analgesic properties.
o BUT also: gastric ulcers, fluid retention and bleeding
Specific COX-2 inhibitors
o Eg. Celecoxib, Rofocoxib
o Anti-inflammatory, antipyretic and analgesic properties- without sideeffects!
o Do not have the anti-thrombotic effect of aspirin
Rheumatoid arthritis (RA)…
Clinical observation of rheumatoid arthritis tells us that there
is prominent destruction of joints (as shown in the adjacent
diagram). The condition seems to improve upon
administration of TNF (tumour necrosis factor) and IL-1
inhibitors.
Very importantly, rheumatoid arthritis is associated with the
presence of rheumatoid factors in the blood.
In rheumatoid arthritis, many individuals produce a group of
auto-antibodies known as rheumatoid factors that are reactive
with determinants in the FC region of IgG.
The classic rheumatoid factor is an IgM antibody with that
reactivity. Such auto-antibodies bind to normal circulating IgG,
forming IgM-IgG complexes which may be deposited in joints.
These immune complexes can activate the complement cascade,
resulting in a type III hypersensitive reaction; leading to chronic
inflammation of the joints.
Above, you can see a question mark next to the labelled antigen. This is because it is
not known whether the antigen must first bind to the IgG, or whether an ordinary
circulating IgG is bound to the rheumatoid factor IgM. This system is therefore
another “hoover” system of the body, and is part of a normal immune response,
however in RA much more of this “hovering” seems to be occurring.
The adjacent diagram shows how immune complexes containing rheumatoid factors
cause disease in RA
In the first response phase, macrophages engulf the IgM-IgG complex with the
antigen bound in most cases. This in turn leads to the
release 2 important cytokines from the synovial
macrophage – IL-1 and TNF. These cytokines lead to the
activation of (cytotoxic) T-cells, and IL-1 is also a
cytokine that is a very important pro-inflammatory
cytokine, thus causes inflammation.
The importance of TNF in RA…
As we already know, TNF is similar to IL-1, but more destructive, i.e. is more
effective in activating T-cells, and so the T-cell response is more potent and
destructive (i.e. destroys foreign antigens more quickly than IL-1), and also leads to
inflammation (i.e. is a pro-inflammatory cytokine).
 In RA, high levels of TNF are present in the joint.
 TNF induces the secretion of metalloproteinases which cause joint destruction.
 Interestingly, TNF transgenic mice also get RA.
 Both human and murine (I think this means something about mice) RA
improves with the use of TNF inhibitors.
Summary of RA…
 First, immune complexes containing rheumatoid factors form in the joint.
 This leads to the activation of synovial macrophages.
 The macrophages engulf the immune complexes and then release TNF and
other pro-inflammatory cytokines.
 TNF (a pro-inflammatory cytokine) induces the secretion of
metalloproteinases; which are known to cause joint destruction.
 The other pro-inflammatory cytokines (such as IL-1 which is also released
from the activated synovial macrophage) also produces inflammation.
Therefore, in RA, the use of TNF inhibitors will prevent the action(s) of TNF
normally released by the synovial macrophages. The main action of the TNF inhibitor
will be to stop the secretion of the metalloproteinases (thus stopping the joint
destruction normally caused); it will also have a secondary role in stopping the
inflammation caused by TNF which is a pro-inflammatory cytokine.
Auto-antibodies…
Auto-antibodies are very important in autoimmune diseases. Firstly, they can be used
in the diagnosis of autoimmune disease, and secondly they can be used to monitor
treatment of autoimmune disease.
Auto-antibodies can be detected using immunofluorescence of the cell or organ
affected (i.e. a broad screening-like test) and also a technique called ELISA or
immunoblotting (which can be used to confirm the specificity of the auto-antibody).
Both of these techniques are discussed in my 4th Term MCD Notes (Laboratory
Diagnostic Methods).
Non-organ specific auto-antibodies are detected by immunofluorescence…
Here, auto-antibodies reacting with the target cell are detected using the method
stated, for example…
 Antinuclear antibodies which may bind to…
– DNA or histones associated with the DNA.
– DNA binding proteins.
– Ribonucleoproteins (important for RNA processing).

Anticytoplasmic antibodies…
– Cytoplasmic ribonucleoproteins.
– ANCAs (antinuclear cytoplasmic antibodies) (in neutrophils).
The antinuclear antibody method…
First, a slide containing fixed cells is taken (shown adjacent).
Next, serum is added which contains the antibody – this binds to the
nuclear antigen.
Then, a fluorescein conjugated anti-Human IgG is introduced (the
thing with the green asterisk thing shown in the adjacent diagram).
Finally, the bound nuclear antigen fluoresces green when the slide is
viewed under UV light microscopy.
Antinuclear antibody staining patterns…
The adjacent diagram shows what you may see with
antinuclear staining…
(A) shows chromosomal staining – this is staining of
the DNA and associated histones.
(B) shows staining of nuclear matrix proteins – this
is staining of RNPs and matrix-DNA for example.
(C) shows staining of the cytoplasm – here staining
is of the RNPs and also cytoplasmic enzymes.
Organ specific auto-antibodies are diagnosed biochemically, for example…
 Diabetes is diagnosed by measuring the (raised) blood sugar levels.
 Thyroid disorders are diagnosed by measuring T4 and TSH levels.
 Pernicious anaemia is diagnosed by measuring haemoglobin levels and Vit B12
levels.
 Antibodies are useful to confirm autoimmune diseases here, but they may also
serve some use in prediction of such diseases (but there is no strong evidence
for this yet).
Non-organ specific antibodies in the diagnosis of SLE and RA…
There is no single diagnostic test to detect for the presence of a certain antibody to
confirm either SLE or RA, that is to say that just because a patient has presence of
antinuclear antibodies (ANA) (as 90% of SLE patients do) this patient has SLE.
Similarly, just because a patient has rheumatoid factors present in a sample (as 80%
of RA patients do) does not mean this patient has RA. You therefore have to look at
the patterns of antibodies and various other factors present in diagnosing either
condition.
Auto-antibodies are used in disease management…
 Some auto-antibody levels fluctuate with disease activity, for example DNA
antibodies in SLE.
 Virtually all other auto-antibodies do not fluctuate with disease activity.
The adjacent diagram shows a treatment
regime used in SLE. I don’t know if you
have to know this in any great detail, but
included it just in case.
The prostaglandin pathway…
The adjacent diagram here simply outlines the
prostaglandin pathway which is important in
generating inflammatory and anti-thrombotic
mediators via the COX-1 and COX-2 enzymes.
Combined COX-1 and COX-2 inhibitors…
Examples of drugs which will do this include: aspirin, ibuprofen, naproxen, and
diclophenac.
 These drugs have anti-inflammatory, antipyretic and analgesic properties.
 However, they may induce gastric ulcers, fluid retention (leading to oedema)
and GI bleeding (mainly via the ulcers they cause).
Specific COX-2 inhibitors…
Examples of drugs which are COX-2 specific inhibitors include: celecoxib and
rofocoxib.
 These drugs have anti-inflammatory, antipyretic and analgesic properties – but
importantly have none of the side effects of combined COX inhibitors.
 However, these drugs do not have the anti-thrombotic effects that are induced
with the administration of aspirin.