Chapter 9: Complement (Complement System)

COMPLEMENT

Complements are essential proteins. These are called complement because it complements the antibacterial activity of some of the antibodies. Compliments are given numerical names as C1 to C9, which are 20 including subunits. Their concentration is 3-4 G/L in the blood.

Complement makes 10% of the serum proteins.

Functions

1. Activation of complements leads to inflammation and localize the antigen or cause lysis.
2. Once complement is activated, its components participate in virtually every aspect of the inflammatory response.
3. There is an antimicrobial activity.
4. Serum sickness-like immune reaction.
5. Autoimmune diseases.
6. These are not increased by infection or other antigens.
7. But IL-1 and γ-interferon increase the synthesis.

Site of Action

The main site is cell surface or other biological membranes.

Changes by Activation of Complement

1. There are ultrastructural changes in the cell membrane.
2. There are changes in the electrical charges of the cell membrane.
3. There is swelling of the membrane.
4. Ultimately there are circular holes 8-12 nm in diameter.
   

   Complement effect on the cell membrane

Site of Formation

1. The complement may be synthesized in the intestinal epithelium, macrophagic cells, and spleen.
2. C1 is a calcium dependant complex and consists of C1q, C1r, and C1s, this is synthesized in the epithelium of the gastrointestinal and urogenital epithelium

Table X – Site of Complement Formation

Complement formation site

Fetus:  Starts forming complement by the second month of pregnancy.

Complement Activation

Complement activation consists of:

1. Recognition of antigen and antibody.
2. Enzymatic chain reaction.
3. Formation of membrane attacking complex which leads to cellular destruction

After Activation Complement is cleaved into:

1. Complements are present in an inactive form in blood circulation, once activated they give chain reaction like blood coagulation factors.
2. Larger molecule labeled as “b”. It leads to further activation of the chain reaction.
3. A smaller molecule labeled as “a” it promotes inflammation and produces pharmacological action.
4. Further proteolysis gives inactive component labeled as “iC3 b”.
5. Complement is catabolized in the body 1-3% per hour.

The following example shows the role of Antibodies and complement in the process of Bacteriolysis and Agglutination.

Fig 69: Role of Complement for Bacteriolysis and Agglutination

Complement pathways

1. Classical pathway
2. Alternative pathway
   

   Fig 68: Complement Pathways

The classical pathway may be activated by:

1. Immunological stimuli-like Ag & Ab complex, tissue injury, or aggregated IgG.
2. non-immunological stimuli like CRP, DNA, Trypsin, E. Coli, Salmonella, viruses, endotoxin, and urate crystals.

Alternative Pathway may be activated by:

1. Immunological stimuli like aggregated IgA, sometimes IgG.
2. Non- immunological stimuli are lipopolysaccharides (endotoxin), Trypsin & trypsin-like enzyme, cobra venom. Parasite & teichoic acid of gram-positive bacteria.

The following diagram gives a summary of the Classical pathway and Alternative pathway.

Fig 70: Summary of Classical and Alternative pathways

Activation of Complement leads to:

1. Lysis of Bacteria, viruses, and cells.
2. Mediate acute inflammation.
3. This leads to the release of histamine.
4. It helps in opsonization and phagocytosis.
5. It has a regulatory role and it regulates acute inflammation in the immune response.
   

   Complement activation and its effect

Various Complement-Proteins lead to:

1. Vasodilatation at the site of inflammation.
2. Increase adherence of phagocytic cells to blood vessels endothelium.
3. It directs movements of phagocytic cells to the area of inflammation.
4. Ultimately clears of the infection.

The outcome of Complement Activation:

1. Physiologic action:
   1. Vasodilatation at the site of inflammation.
   2. Increased vascular permeability.
2. Cellular action:
   1. Increase adherence of phagocytic cells to blood vessels endothelium.
   2. Recruitment of acute inflammatory cells.
   3. It directs movements of phagocytic cells to the area of inflammation.
   4. Produce inflammatory mediators.
   5. Hemolysis in the case of RBCs.
   6. Cytolysis.
   7. Opsonization of the pathogens.
      1. Ultimately clears of the infection.
      2. Ultimately killing of pathogens.

Fig 70 A: Outcome of Complement activation

Complement Receptors

Various cell types express surface membrane glycoproteins that react with one or more of the fragments of C3 produced during complement activation and degradation.

Complement receptor 1 (CR1) is important in increasing phagocytosis. CR1 is an important factor present on the RBCs.

CLASSICAL PATHWAY ACTIVATION

Overview

Suppose this reaction is taking place on the cell membrane of the RBC and the sequence of the event may be as follows:

Fig 71: Complement action on the RBC

This can also be explained in the following diagram in a more simple way.

Fig 72: Classical Complement Pathway

In the classic pathway antigen and antibody complex is recognized by the complement (C1q) and it starts a chain reaction by stimulating C1s and C1r which will stimulate C4 and C2. Then there is the stimulation of C3 C5 C6, 7, and ultimately C9.

This will give rise to MAC  (membrane attacking complex) and leads to the osmotic death of the target cells.

Details of Classical Pathway Activation

Now the details of the classical pathway can be elaborated when suppose the reaction is occurring on the cell membrane where there is an antigenic determinant (Epitope).

The activation of complement is not in sequence from C1 to C9, but this is like this:

C1,  4, 2, 3, 5, 6, 7, 8 and 9.

Fig 73 Classical pathway activation

The sequence of activation of the classical complement pathway:

1. C1q is the recognition unite and it recognizes the Ag-Ab complex and then activates C1r.
   1. 1. C1q directly binds to the immunoglobulin molecule.
      2. C1r and C1s do not bind the immunoglobulin but leads to the subsequent activation of C3.
      3. C1q only attaches to subclasses of IgG and IgM.
      4. IgM single molecule is able to fic C1 but IgG needs two molecules.
      5. C1q is functionally multivalent for to attach to the complement fixation sites of immunoglobulin.
      6. C1r and C1s are similar in structure, where C1r forms dimers and C1s binds to C1r.
      7. Now, C1s and C1q both bind to C1q in the presence of Ca⁺⁺.
      8. C1s is the only substrate for C1r.
      9. C1r^– cleaves C1S (C1S^–).
   2. C1s cleaves C4 to C4b (large components)  and C4a.
      1. This function takes place in the fluid phase of the plasma around the C1s.
      2. C1s are weekly proteolytic for free C2 but highly active against C2 that has complexed with C4b molecule in the presence of Mg⁺⁺ ions.
      3. C1s also cleaves C2 to C2a and C2b.
      4. This reaction takes place when the C4bC2 complex is near the C1s.
      5. The control mechanism for the activity of C1s is a C1-proteinase inhibitor, which will control the C1s for un-controlled activation of C4 and C2.
         1. C3b esterase inhibitor has the ability to disintegrate the membrane-bound C4b.
         2. This action destroys the acceptor site for C2, and this will prevent the formation of the C4bC2b convertase complex.
2. C4b molecule has a stable binding to the cell membrane (efficiency is <10%).
   1. C4b molecule which can not attach to the cell membrane will become inactive and decay.
   2. The resulting C2b joins the C4b and forms the complex C4bC2b which is an enzyme.
   3. C4bC2b complex enzyme is unstable and decays with a half-life of 5 minutes at 37 °C, due to the release and decay of C2b.
   4. C4b and C2b act as C3-convertase and cleaves C3 to C3a and  C3b.
      1. C4bC2b convertase will activate the C3 molecule into C3a anaphylatoxin and C3b.
3. C3b: Clusters of C3b are activated and bound near the C4bC2b complex.
   1. Each catalytic site can bind several hundreds of C3b molecules.
   2. Only a C3b molecule combines with C4bC2b to form the final proteolytic complex complement pathway.
   3. The complex of C4bC2bC3b forms an opsonic macromolecular coat on the target cells and makes it susceptible to immune adherence by the C3b receptor on the phagocytic cells.
4. C4b C2b C3b acts as a C5 convertase and cleaves C5 into C5a and C5b.
5. C5b fixation and is the beginning of the membrane attack complex.
   1. No further proteinases are generated in the classical complement pathway.
   2. C5b binds to C6 and make a labile reactive site for C7.
6. C5b 6, 7 is highly lipophilic and binds to the membrane where it acts high-affinity receptors for C8.
   1. In free solution, uncombined C567 has a half-life of about 0.1 seconds.
   2. It can attach to any lipid layers within its effective diffusion radius and will produce the picture of reactive lysis on innocent bystanders cells.
   3. Membrane-bound C567 is stable and can interact with C8 and C9.
7. C8 has three chains α, β, and γ where γ-chains insert into the membrane.
8. C5b678 polymerizes C9 forming tubule the MAC (membrane attacking complex), which gives rise to hole formation in the cell-membrane Like drill machine.
   1. This tubule is a hollow cylinder with one end inserted into the lipid layer and other ends can project from the membrane.
   2. C5b678 makes a 3nm hole and cells become leaky.
   3. C5b6789 makes transmembrane holes, 15-16nm long and 8-12nm in diameter. This leads to the passage of electrolytes and water and causes osmotic lysis (death).
   4. By combing with C9, the cytolytic reaction is accelerated.
9. This hole formation can disturb the lipid layer and allow the free exchange of ions as well as water across the membrane.
   • The end result is in the living cell is an influx of Na⁺ and H2O leads to disruption of the osmotic balance which produces cell lysis.

Functions of Various Components:

1. C1q
   1. It is evaluated in the serum.
   2. It is needed in the first reaction of complement where it will lead to activation of C4 and C2.
2. C2 and C2a
   1. It takes part in the activation of C3 with the help of C4.
   2. It increases vascular permeability.
   3. It causes contraction of smooth muscles.
   4. Some say that functions are unknown.
3. C4a
   1. It is a weak anaphylatoxin and weak mediator of Inflammation.
4. C3a
   1. It is a strong anaphylatoxin.
   2. It leads to an increase in vascular permeability and smooth muscle contraction.
   3. This leads to the release of vasoactive amines.
   4. Leads to the release of lysosomal enzyme.
   5. It is chemotactic.
5. C5a
   1. Functions just like C3a.
   2. It increases neutrophil activity.
   3. More potent chemotactic factor than C3a.
6. C6
   1. Activated by the C5b and attach to C7.

Anaphylatoxin

1. These are C3a, C5a, and C4a.
2. This name is because of their role in anaphylaxis, where these directly activate mast cells and basophils, through their receptor for C5a, and lesser extent C3a.
3. The anaphylatoxin activity is:
   1. Smooth muscle contraction.
   2. Vasodilatation.
   3. Increases vascular permeability.
   4. Give rise to the release of histamine.
   5. These are chemotactic agents.
   6. These initiate the inflammation.
   7. These can generate cytotoxic oxygen radicals.
4. Their main function is degranulation of the mast cells or basophils, endothelial cells, and phagocytes.
   1. C5a has the highest biologic activity.
   2. C3a acts with C5a to activate mast cells, recruit antibody, complement, phagocytes, and increase the tissue fluid in that area.
   3. C4a has the least anaphylactic activity.
      

      Fig 74: Mast cell and Basophil C3a and C5a receptors

Complement components and their functions:

Table XI – Complement proteins

Check Mechanism or Inhibitors of the Classical Pathway:

Nature has given a check mechanism, which controls unlimited damage caused by the activated complement system even to the innocent bystander cells. These inhibitors are:

1. C1-inhibitor: It is a glycoprotein and also known as C1 esterase. It destroys C1r, C1s.
2. The factor I:  It is proteolytic enzymes, it cleaves C3b into iC3b (inactive form). It also destroys C4b.
3. Vitronectin (S-protein): Prevents binding of C5, 6, 7 to the membrane.
4. Decay Accelerating Factor (DAF): Prevents activation of C4.
5. C4 binding protein: Prevents activation of C4.
6. Factor H: It binds C3b and accelerates the destructive action of factor I.

ALTERNATIVE PATHWAY

1. This is a primitive defense system. In this pathway, there is a bypass of C1, 4, 2 and there is direct stimulation of C3.
2. This is predominantly a non-antibody initiated pathway.
3. The activator of the alternative complement pathway are:
   1. Polysaccharides complex from the surface yeast cells.
   2. Bacterial polysaccharides and endotoxin.
   3. Aggregated immunoglobulins IgG2, IgA, and IgE.
   4. Zymosan.
   5. Inulin.
4. The nonspecific activation is a major physiologic advantage because the host protection can be generated before the induction of a humoral immune response.
5. C3a is considered the counterpart of the C2a of the classical pathway.
   1. C2 of the classical pathway resembles factor B of the alternative pathway.
6. There is no role of C1, C2, and C4, but the alternative pathways have different protein which stimulates directly C3.
7. Factor C3b and factor B combine to form C3b, B, which is then converted into C3 convertase (C3bBb).
   1. Factor Ba is a glycine richα-2-globulin believed to be physiologically inactive through the action of factor D.
   2. C3b, Bb complex can convert more C3 to C3b, in turn, it binds more factor B.

Fig 75: Alternative Complement pathway

After the stimulation of C3 then there is a common pathway with the help of factor B and factor D. In the end, there is the formation of MAC (membrane attacking complex).

Alternative complement pathway and their components:

Table XII – Proteins of the alternative pathway

Positive Regulators for Alternative Pathways are certain substances, which enhance and give positive regulation of the alternative pathways. These are:

1. Factor P (Properdin).
2. Cobra Venom Factor (CoVF):- This complexes with Bb (CoVFBb) which acts as C3/C5 convertase. It is resistant to factor H & I.

Inhibitors of Alternative Pathways

These are natural check system or inhibitors.

1. Factor H which binds to C3b.
   1. Factor H prevents the association of C3b and factor B.
   2. Factor H block the formation of C3b, Bb complex.
   3. Factor H competes with factor B for its combining sites on C3b and ultimately leads to the inactivation of C3 convertase.
   4. factor B and H occupy a common site on C3b.
   5. Polysaccharides are called surface activators and favor the uptake of factor B on the chain of C3b, and it will displace the factor H.
   6. The other controlling point is the amplification of C3b, Bb depends upon the stability of C3b-Bb convertase. C3b -Bb decay due to loss of Bb which has a half-life of roughly 5 minutes.
   7. If factor P combines with C3b-Bb and form C3b-Bb-P, then the half-life is increased to 30 minutes.
   8. The factor I which inactivate and cleaves C3b.

Complement Activation is an Amplification Process.

1. Roughly 1200 C3b opsonin can deposit with a single molecule of IgM.
2. A single IgM molecule cleaves one C1q and C1s and these will cleave 100 C4 molecules.
3. 20 or 50 C4b cleaves about 100 C2 molecules.
4. Many C5 molecules initiate MAC (membrane-attacking complex).

Complement Receptors

The most important function of complement is to facilitate the uptake and destruction of the pathogen by phagocytic cells. This occurs by specific recognition of bound complement components by complement receptors (CR) present on phagocytic cells.

Similar complement receptors are present on RBC and RBC play an important role in the clearance of soluble Ag + Ab complexes from the circulation by carrying these (Ag + Ab) complexes to a reticuloendothelial system like the spleen.

Table XIII – Receptors present on various cells

Complement receptors and their functions:

Table XIV – Various receptors

Biological Functions of Complements:

Human Diseases Associated with Complement Deficiency:

Deficiency of any component of complement leads to various diseases e.g.

Raised level of complement:

1. Inflammatory process.
2. Trauma.
3. Myocardial infarction.
4. In acute illnesses where complement raised as acute-phase proteins.

Decreased level of complement (deficiency of complements):

1. Autoimmune diseases like:
   1. SLE.
   2. Rheumatoid arthritis.
   3. Systemic vasculitis.
   4. Essential mixed cryoglobulinemia.
2. Infectious diseases:
   1. Arterioventricular shunts with infection.
   2. Subacute bacterial endocarditis.
   3. Gram-negative sepsis.
   4. Pneumococcal sepsis.
   5. Viral hepatitis B leading to viremia.
   6. Viremia in measles.
   7. Malarial parasite infestation.
3. Deficiency of controlling proteins:
   1. These are quite uncommon primary immune deficiency <2%.
   2. The factor H deficiency.
   3. The factor I deficiency.
   4. Hereditary angioedema (C1 inhibitor deficiency).
      

      Deficiency of complements and the diseases caused by:

      Deficiency of complement	Diseases caused by the complement deficiency
      C1s	SLE Angioedema
      C1r	Glomerulonephritis Chronic infection SLE Dermatomyositis Vasculitis Necrotizing skin lesion Arthritis
      C1q	SLE Decreased secondary to agammaglobulinemia
      C2	Recurrent pyogenic infection SLE Membranoproliferative glomerulonephritis Discoid lupus erythematosus Dermatitis herpetiformis Dermatomyositis Synovitis Purpura Hodgkin’s disease Chronic lymphocytic leukemia Polymyositis
      C3	Recurrent pyogenic infection SLE Arthritis Skin rash
      C3b inactivator	Recurrent pyogenic infection Urticaria
      C4	SLE Vasculitis Dermatomyositis like syndrome
      C5	Recurrent infection (Neisserai) SLE
      C6	Gonorrheal infection Meningococcal infection SLE Scleroderma Vasculitis Raynaud’s phenomenon
      C7	Raynaud’s disease Renal disease Neisseria infection SLE Vasculitis Scleroderma
      C8	Glomerulonephritis SLE Neisseria infection Xeroderma pigmentosa

Complement Fixation Test

The complement may be used as a diagnostic tool. The basic principle is its activity which leads to hemolysis of RBC. In this test, we need:

1. Serum of the patients.
2. Complement
3. Indicator system which consists of antibody-coated RBC.
4. Known antigen.

This test is used to find Unknown Antibody or Antigen.  The following diagram shows the basic mechanism.

Fig 76: complement based test

This test may be manipulated by making dilution of serum or complement or one can have known antibody and unknown antigen or vice versa.  Even the titer of antibody or concentration of complement may be estimated by serial dilution.

Use of Complement fixation test

1. This test can be used to diagnose various diseases e.g Pneumococcal pneumonia, syphilis and etc.
2. With the help of this test, one can quantitate antigen or antibody.
3. This test was very famous for the diagnosis of syphilis.