Angioedema: Part 4

Deficiencies of an early component of the classical complement pathway (C1q, C1r, C1s, C2, C4) have been associated with lupus like autoimmune conditions. The reason for this is that these proteins help to clean up large groups of molecules called immune complexes before they can cause inflammation. Dead cells are also removed by these complement molecules. Without these proteins, immune complexes and dead cells are not removed and cause local irritation.

In HAE types I and II, complement proteins C2 and C4 are low. However, HAE patients have been shown to have a normal level of immune complexes. For this reason, it is still unclear whether or not low C2 and C4 may contribute to overall inflammation and pain profile for these patients. Despite this fact, it is still possible that deficiency in C2 and C4 may predispose HAE patients to autoimmune diseases.

A number of studies have assessed the prevalence of autoimmune conditions in HAE patients. One study looked specifically for two thyroid antibodies and found that 13.2% HAE patients had autoantibodies to the thyroid.

When expanding the autoimmune profile to include “lupus-like” conditions such as those often associated with complement deficiencies, a much higher prevalence of autoantibodies was found in HAE patients. Three other studies measured the frequency of ANA (anti-nuclear antibody, a generic marker found in many autoimmune conditions); RF (Rheumatoid Factor, associated with rheumatoid arthritis); anti-thyroglobulin(autoimmune thyroiditis); TPO (thyroid peroxidase, autoimmune thyroiditis); and thyroid antibodies along with some or all of the following antibodies: anti-dsDNA (anti double stranded DNA, systemic lupus erythematosus); ENA (extractable nuclear antigens, a panel of six tests that can identify mixed connective tissue disease, systemic lupus erythematosus, Sjogren’s, Scleroderma and dermatomyositis); TMA (microsomal antibodies, autoimmune thyroiditis); AMA (antimitochondrial antibodies, drug-induced or systemic lupus erythematosus, Sjogren’s, autoimmune hemolytic anemia, autoimmune liver disease); ANCA (antineutrophil cytoplasmic antibodies); anti-cardiolipin (systemic lupus erythematosus, Behcet’s, antiphospholipid syndrome); anti-b2GPI (b2-glycoprotein I, systemic lupus erythematosus, Behcet’s, antiphospholipid syndrome); anti-C1q (urticarial vasculitis); anti-P ribosomal (systemic lupus erythmatosis); EMA (anti-endomysial antibodies, Celiac disease); tTG (anti-tissue transglutaminase antibodies, dermatitis herpetiformis); and ASCA (anti-saccharomyces cerevisiae antibodies, Behcet’s, Celiac disease, Crohn’s disease, ulcerative colitis). The three studies found that 47.5-48% HAE patients had at least one of these autoantibodies. In comparison, the average for healthy controls was 10%.

Other studies looked at prevalence of autoimmune disease rather than autoantibodies. One study found that 12% of HAE patients had at one of the following autoimmune conditions: glomerulonephritis, Sjogren’s syndrome, irritable bowel disease, thyroiditis, systemic lupus erythematosus, rheumatoid arthritis, drug induced lupus, pernicious anemia, juvenile RA with IgA deficiency, or sicca syndrome.

Other studies found that 3.4% HAE patients had lupus rash or glomerulonephritis; that 0.9% had RA or Sjogren’s; that 11.5% had Crohn’s, Celiac, Hashimoto’s thyroiditis, discoid lupus erythematosus, chronic lymphocytic leukemia, MGUS, or IgA deficiency; that 11.4% had systemic lupus erythematosus, Celiac, multiple sclerosis-like syndrome, systemic sclerosis, or mixed connective tissue disease; that 4.2% had lupus like syndrome, psoriatic arthritis, mixed connective tissue disease or antiphospholipid syndrome; that 0.4-0.9% had lupus-like or unspecific cutaneous lupus or subacute lupus.

An interesting feature of HAE is the frequent complaint of decreased sense of smell. Facial edema and chronic rhinosinusitis were not found to be the cause. However, systemic lupus erythematosus and Sjogren’s syndrome can also cause impairment of smell. Despite the frequency of lupus in HAE patients, it usually affected the mucocutaneous regions of the body and was generally mild.

In addition to the frequent prevalence of autoantibodies and autoimmune disease, HAE patients have increased B cell activation and autoreactive B cells. This can also contribute to an inflammatory and autoimmune profile.

 

References:

Kaplan AP, et al. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammation pathway. Adv Immunol 2014; 121:41-89.

Kaplan AP, et al. The plasma bradykinin-forming pathways and its interrelationships with complement. Mol Immunol 2010 Aug; 47(13):2161-9.

Firinu, Davide, et al. Characterization of patients with angioedema without wheals: the importance of F12 gene screening. Clinical Immunology (2015) 157, 239-248.

Csuka, Dorottya, et al. Activation of the ficolin-lectin pathway during attacks of hereditary angioedema. J Allergy Clin Immunol 134 (6) 1388-1393.e3.

Triggianese, Paola, et al. The autoimmune side of hereditary angioedema: insights on the pathogenesis. Autoimmunity Reviews 2015 (ahead of press).

 

 

 

 

Activating the complement system: Classical, alternative and lectin pathways

The complement system is part of the innate immune system, meaning it does not “learn” over time by being exposed to organisms, and its behavior is the same throughout life. This system is made up of many small proteins that are manufactured in the liver and then released into the bloodstream. Importantly, these proteins are in an inactive state when they move out of the liver. To be active, these proteins have to be “cleaved” or have pieces of the molecule cut off. This is done by other proteins when specific signals are detected.

The complement system undergoes large scale amplification, meaning many, many proteins can be cleaved to fight infection from only one small signal. Once there are many complement proteins to help, they help to kill microbes by building a tunnel through the cell membrane. This tunnel is called the membrane attack complex (MAC).

There are three methods for activating the complement system, all of which involve several steps and several molecules.  It is crucial that these complements are present in the correct ratio or it can contribute to inflammation and disease.

The classical pathway is activated in one of three ways:

  1. Activation:
  • An antibody binds to the outside of microbe. This can be done by certain types of IgG (but not IgG4) or IgM.
  • The enzyme C1 can also bind to the surface of some microbes.
  • C-reactive protein can also activate the classical system by binding to some microbial products.
  1. In the blood, C1 is actually made up of three small parts called subunits: C1q, C1r and C1s. The C1q binds to the antibody on the surface of the microbe. This activates the subunits.
  2. C1s cleaves C4 into two pieces. C4b binds to the cell surface of the microbes. C4a has no function here and is broken down after being released.
  3. C1s cleaves C2 into two pieces. C2b binds to C4b, which is bound to the cell surface. C2a has no function here and is broken down after being released.
  4. When C4b and C2b are bound together, they are called C3 convertase and they perform the special function of cleaves C3. C3 is cleaved into two pieces.
  5. C3b binds to various places on the cell surface. Macrophages and neutrophils (immune cells) can bind to C3b. When macrophages bind to C3b, it may then phagocytose (or eat) the microbe. C3b can also bind to C5, which allows it to be cleaved by C3/C5 convertase.
  6. C3a is an anaphylatoxin. (I have written a previous detailed post on this). It can trigger basophils and mast cells to degranulate.
  7. C5 is cleaved by C3/C5 convertase. This release C5a and C5b.
  8. C5a is a very strong anaphylatoxin and also attracts neutrophils to fight infections.
  9. C5b is the anchor for the membrane attack complex. C6, C7, C8 and several molecules of C9 form a long line on molecules that pokes a whole in the membrane of the microbe. If the membrane is broken, water will rush into the cell and the cell will not function correctly. This results in cell death.

The alternative pathway is activated as follows:

  1. C3 can turn itself into the molecule C3b. This is spontaneous and does not require any other molecules. C3b is short lived under normal circumstances.
  2. If a microbe is nearby, C3b will bind to a molecule on the microbial surface called Factor B.
  3. C3b and Factor B bound together are a different kind of C3 convertase than the one described for classical pathway. This C3 convertase cleaves other molecules.
  4. C3b-Factor B, a C3 convertase, cleaves a molecule of C3.
  5. The liberated molecule of C3b binds to C3b-Factor B-C3b. This is a C5 convertase, which starts the membrane attack complex.
  6. While the MAC is being made, this C5 convertase is still cleaving C3 to release large amounts of C3b.

The lectin pathway is activated as follows:

  1. MBL and ficolin bind to microbial surfaces.
  2. This activates the molecule MASP-2.
  3. MASP-2 cleaves C4 and C2, forming a grouping of molecules called the terminal complement complex (TCC).
  4. C1s cleaves C2 into two pieces. C2b binds to C4b, which is bound to the cell surface. C2a has no function here and is broken down after being released.
  5. When C4b and C2b are bound together, they are called C3 convertase and they perform the special function of cleaves C3. C3 is cleaved into two pieces.
  6. C3b binds to various places on the cell surface. Macrophages and neutrophils (immune cells) can bind to C3b. When macrophages bind to C3b, it may then phagocytose (or eat) the microbe. C3b can also bind to C5, which allows it to be cleaved by C3/C5 convertase.
  7. C3a is an anaphylatoxin. (I have written a previous detailed post on this). It can trigger basophils and mast cells to degranulate.
  8. C5 is cleaved by C3/C5 convertase. This release C5a and C5b.
  9. C5a is a very strong anaphylatoxin and also attracts neutrophils to fight infections.
  10. C5b is the anchor for the membrane attack complex. C6, C7, C8 and several molecules of C9 form a long line on molecules that pokes a whole in the membrane of the microbe. If the membrane is broken, water will rush into the cell and the cell will not function correctly. This results in cell death.

 

Some molecules control the complement system so that the amplification does not cause problems.

  • Factor H controls the alternative pathway. It helps to degrade the C3b-Factor B-C3b complex.
  • Factor I converts C3b to an inactive form.
  • C1INH (C1 inhibitor) binds to activated C1r and C1s, making them inactive. This happens quickly, so there is only a brief time before C1INH binds to C1r or C1s during which they can cleave C4 and C2.

 

 

 

Chronic urticaria and angioedema: Part 3

There are several pathways that can culminate in angioedema and urticaria.

Activation of mast cells by IgE is the most well known mechanism. When IgE binds to receptors on mast cells, several things happen. The mast cells release histamine. This in turn causes dilation of the nearby vessels and causes fluid to leak from the bloodstream into the tissues. This causes nerve cells to activate and release substance P, which also contributes to vasodilation and causes mast cells to release more histamine. In response to activation by IgE, mast cells will also produce PGD2 and leukotrienes C4 and D4.

The complement system is one of the ways our body identifies infectious agents and triggers the immune system to kill them. Complement proteins are in the blood all the time, and they can be activated by three distinct pathways, all of which are triggered by pathogens: the classical pathway, the alternative pathway and the lectin pathway. Regardless of which pathway activates the complement system, the molecules C3a, C4a and C5a are produced. These molecules bind to receptors on mast cells and induce histamine release.

Following initial dilation of local vessels, proteins that normally are found in the plasma move into the skin. This activates the kinin system, which produces bradykinin through a series of steps. Bradykinin is a very powerful vasodilator and contributes significantly to loss of volume from the blood stream to the tissues.

C3a, C5a, PGD2, and leukotrienes C4 and D4 all draw other inflammatory cells to the site of activated mast cells. These cells release further molecules to stimulate histamine release. This mechanism perpetuates inflammation beyond the original insult.

Bradykinin levels are normally controlled by the enzyme ACE. When patients take ACE inhibitor medications (like Lisinopril, etc), this interferes with bradykinin degradation and cause urticarial and angioedema.

C1 esterase inhibitor regulates complement and kinin pathways. In patients who are deficient in C1 esterase inhibitor, bradykinin may be overproduced.

Many autoimmune conditions cause the formation of IgG1 and IgG3 antibodies. These molecules can interfere with the complement system and cause production of fragments that activate mast cells, like C3a.

NSAIDs are well characterized in their ability to cause angioedema and urticaria. While the mechanism is not fully understood, it is thought that since NSAIDs stop production of prostaglandins, the mast cells overproduce leukotrienes, which contribute to the angioedema and urticaria.

There are several non-immunologic methods that can result in angioedema and urticaria. Heat or pressure on the skin; radiocontrast dyes; alcohol; vancomycin; opioids; and foods like shellfish and strawberries have been linked to these conditions.

 

References:

Jonathan A. Bernstein, et al. The diagnosis and management of acute and chronic urticaria: 2014 update. J Allergy Clin Immunol Volume 133, Number 5.

Usmani N,Wilkinson SM. Allergic skin disease: investigation of both immediate and delayed-type hypersensitivity is essential. Clin Exp Allergy 2007;37:1541-6.

Zuberbier T, Maurer M. Urticaria: current opinions about etiology, diagnosis and therapy. Acta Derm Venereol 2007;87:196-205.

Ferdman, Ronald M. Urticaria and angioedema. Clin Ped Emerg Med2007; 8:72-80.

 

How to activate mast cells: Complement protein C3a

The complement system is part of the innate immune system. It is composed of many small proteins that circulate as inactive precursor molecules. When the immune system is triggered, enzymes cleave these precursors to form activate cytokines. These cytokines then cleave other precursors to form even more cytokines. This is known as a cascade and it amplifies the inflammatory signal to draw other inflammatory cells and molecules.

Mast cells express many receptors on their surfaces. The one people are most familiar with is the IgE receptor, also called FceRI. One of the receptors mast cells express is for C3a, a fragment of complement protein C3. This fragment is produced by the complement activation cascade. C3a is known for inducing smooth muscle contraction, increasing vascular permeability, recruiting white blood cells to the site of skin injection, and attracting macrophages, neutrophils, some lymphocytes, basophils and mast cells.

Something I haven’t touched on much is the fact that there are two major categories of mast cells in the body. Mucosal mast cells live in the GI mucosa. Serosal mast cells live in the skin, peritoneum and respiratory tract. These two populations have different mediators in their granules and respond differently to stimuli like C3a. In mucosal mast cells, C3a actually inhibits histamine and TNF release. In serosal mast cells, C3a increases degranulation of cells stimulated by IgE or IgG.

C3a has also been shown to cause expression of particular genes in mast cells that participate in production of cytokines. This is achieved by multiple pathways, one that works at low concentrations of C3a (cell surface GPCR) and one that works at higher concentrations (activation of G proteins).

In patients with allergic asthma, inhaled allergens like dust, dust mites, Aspergillus and ragweed pollen, activate the complement system in the mucosa of the respiratory tract. This generates the formation of C3a. Cells around mast cells release enzymes that can cleave C3 to form C3a. When mast cells become activated, they release a number of enzymes that may also cleave C3 to form C3a. Tryptase has been shown to do this in vitro, meaning in a reaction outside of the body.

When some receptors are stimulated too often, they become desensitized. This causes a signal to be sent into the cell that makes the cell internalize the receptor, or literally remove it from the surface so it can’t be activated anymore. This is the case for mast cell C3a receptors. I am curious to know if C3a receptors in mast cell patients don’t get desensitized. In theory, this would result in huge, fast allergic reactions without IgE stimulation and chronic activation of the inflammatory response. This has not been investigated to my knowledge.

Several mast cell mediators, including histamine, make blood vessels more permeable. Some researchers hypothesize that this action works to draw C3 to the activated mast cells. C3 can then be cleaved by tryptase, producing C3a, and amplifying the allergic reaction. Due to its well characterized role in anaphylaxis and allergic response, C3a is known as an anaphylatoxin.

 

References:

Erdei et al. Regulation of mast cell activation by complement-derived peptides. Immunology Letters 92 (2004) 39–42.

Ali H. Regulation of human mast cell and basophil function by anaphylatoxins C3a and C5a. Immunology Letters 128 (2010) 36–45.

M.R. Woolhiser, K. Brockow, D.D. Metcalfe. Activation of human mast cells by aggregated IgG through FcγRI: additive effects of C3a, Clin. Immunol. 110 (2004) 172–180.

T.C. Theoharides et al. Mast cells and inflammation. Biochimica et Biophysica Acta 1822 (2012) 21–33.