Conclusiones del Marco Conceptual

‰ IMMUNE SYSTEM

As white cells play a major role in immunity, it is appropriate to consider antibodies and complement here.

Antibodies

Antibodies are immunoglobulins that react with antigens. They are produced by plasma cells, which in turn are derived from B lymphocytes.

Structure of Immunoglobulins

The immunoglobulin molecule consists of two identical heavy (H) chains and two identical light (L) chains. The H and L chains are linked together by disulfide (s-s) bonds. Five classes of immunoglobulins are recognised based on the type of H chain:

IgA (α or alpha H chain), IgD (δ or delta), IgE (ε or epsilon) IgG (γ or gamma), and IgM (µ or mu). Light chains are of two varieties—κ (kappa) and λ (lambda).

A molecule of immunoglobulin consists of light chains of the same type (either κ or λ); both types of light chains are never present together. Kappa and lambda chains are present in 2:1 proportion in immunoglobulins.

Each chain has a constant and a variable region (Fig. 1.23). Amino acid composition in the carboxy terminal region of heavy chain and light chain is the constant region;

in the heavy chain it determines the class of the immunoglobulin molecule. The CH2 domain in IgG binds complement while CH3 domain binds to Fc receptor of monocytes.

The variable region of the molecule (VL and VH) is the specific antigen-binding site and is in the amino-terminal part of the molecule. The area J of the heavy chains in the constant regions between CH1 and CH2 domains is flexible and is called hinge region;

due to this the two antigen-binding sites can move in relation to each other spanning variable distances.

Each immunoglobulin molecule can be digested by a proteolytic enzyme papain just above the disulphide bond joining the two heavy chains into three parts: one Fc and two Fab fragments. The fragment, which contains the carboxy terminal and constant parts of both heavy chains, is called the Fc (fragment crystallizable) fragment. Each Fab (fragment antigen binding) fragment contains amino terminal portion of H chain and complete light chain and has the antigen-combining site (Fig. 1.23).

Figure 1.23: Structure of immunoglobulin molecule. Broken line indicates site of papain digestion

circulating immunoglobulins. IgG is the monomer of the basic immunoglobulin structure. There are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. Relative concentration in serum can be represented as IgG1 >lgG2>lgG3>lgG4. IgG is usually produced during secondary immune response. It is the only immunoglobulin, which is transferred transplacentally to the foetus from the mother. The foetus cannot synthesize IgG and therefore IgG antibodies in the newborn represent those passively gained from the mother. IgG is capable of fixing complement with order of efficacy being IgG3, IgG1 and IgG2. IgG4 cannot bind complement in the classical pathway.

Only IgG3 and IgG 1 can bind to Fc receptors on macrophages.

IgM: This has high molecular weight and is also called as macroglobulin due to its large size. IgM molecules have a pentameric structure (i.e. five immunoglobulin units joined together) and also have an additional short polypeptide chain (J or joining chain). It comprises 5–10% of circulating immunoglobulins. IgM is the first antibody produced in response to the antigen (primary response). In contrast to IgG, IgM cannot cross the placenta. The foetus is able to produce IgM after maturation of its immune system.

IgM is highly efficient in binding complement. A single molecule of IgM can bind complement while two molecules of IgG (lgG doublets) are necessary for complement-binding. The order of efficiency of complement binding of immunoglobulins is IgM, IgG3, IgG1 and IgG2. There are no receptors on macrophages for IgM.

IgA: There are two subclasses of IgA: IgA1 and IgA2. IgA is present mostly in body secretions such as gastrointestinal and respiratory mucosal secretions, saliva, tears, etc. Secretory IgA is mostly IgA2 and exists as a dimer. Serum IgA, which is mostly IgA1, is a monomer.

IgD and IgE: Both are present in trace amounts in serum and are monomeric.

Most IgD is expressed on the surface of resting B lymphocytes where it serves as an antigen receptor.

Most IgE is bound to basophils or mast cells through heavy chain. When a specific antigen combines with IgE, vasoactive substances are released from these cells and lead to anaphylaxis.

Alloantibodies vs autoantibodies: Alloantibodies are those which are produced by an individual against antigens present in another individual of the same species.

Autoantibodies are those, which are produced by an individual against one’s own antigens.

Warm vs cold antibodies: Warm antibodies react maximally at 37°C while cold antibodies show maximum activity at 0 to 4°C. Most IgG antibodies are of warm type while most IgM antibodies are of cold type. Characteristic features of different immunoglobulins are presented in Table 1.3.

Complement

Complement are serum proteins which when activated react in an orderly manner with each other to cause immunologic destruction of target cells (lysis or phagocytosis).

There are three pathways of complement activation: classical, alternate and mannose-binding pathway (Fig. 1.24).

Classical Pathway

Classical pathway is usually initiated by reaction of antibody (IgG or IgM) with antigen (e.g. red cells). Binding of only a single IgM pentameric molecule or of IgG doublet to an antigen are necessary for complement activation.

The complements are activated in the following order: Ag-Ab complex—C1 C4 C2 C3 C5 C6 C7 C8 C9. This process occurs on the surface of target cells (e.g. red cells). Binding of antibody to antigen causes exposure of complement-binding site on immunoglobulin.

The activated C1 cleaves C4 to form C4a and C4b; C4a is released into the body fluid while C4b attaches to the red cell membrane. Activated C1 also cleaves C2 to form C2a. The C4b2a complex (C3 convertase) is formed. The C4b2a complex attached to cell membrane has enzymatic activity and can cleave several hundred C3 molecules. The C3a is released into plasma while C3b attaches to the cell membrane. C3b however is rapidly degraded into C3dg. C3b is not enzymatically active by itself, but presence of C3b on the cell surface is recognized by specific receptors on the surface of macrophages and this causes phagocytosis of C3b-bearing cells. C3dg cannot adhere to macrophages because macrophages do not have receptors for C3dg. Once C3b is converted to C3dg, then complement cascade is terminated; C3dg coated red cells in circulation are resistant to further complement-mediated cell destruction.

Some C3b joins C4b2a to form C4b2a3b (C5 convertase). C5 convertase cleaves C5 into C5a and C5b. C5a is released in circulation. C5b joins with C6 C7 C8 C9 to form membrane attack complex (MAC), which fixes on cell membranes and causes cell lysis.

The MAC creates pores in red cell membrane through which water enters into red cells, cells swell and are lysed.

Table 1.3: Characteristics of immunoglobulins

Parameter IgM IgG IgA IgE IgD

1. Approx % of total Ig 5% 80% 15% Trace Trace

2. Molecular weight 900,000 150,000 150,000 or 300,000 190,000 180,000

3. Heavy chain µ γ α ε δ

4. Structure Pentamer Monomer Dimer (secretions),

monomer (serum)

Monomer Monomer

5. Half-life (days) 5 21 6 2 3

6. Complement activation Yes Yes No No No

7. Placental transfer No Yes No No No

8. Main function Primary immune response

Alternate Pathway

In alternate pathway, C3 is activated directly with no role of earlier complement components. It does not require antigen-antibody reaction.

C3 can be activated by endotoxins, complex carbohydrates such as are present on some micro-organisms, and aggregates of IgA. A serum protein called properdin, factors B and D, and magnesium ions are needed for activation of alternate pathway.

Normally, C3 is being continuously cleaved at low level, probably by factor B, resulting C3b is rapidly cleared from the plasma. However, when C3b comes in contact with certain substances (e.g. complex carbohydrates on the surface of micro-organisms) then association of C3bB occurs on the surface of micro-organisms in the presence of Mg⁺⁺ ions. Factor B is cleaved by factor D to form C3bBb. Properdin may stabilise C3bBb.

C3bBb splits C3 to generate more C3b thus forming an amplification loop.

Alternate pathway plays an important role in initial defense against infection in nonimmune persons.

Mannose-binding lectin pathway: Mannose-binding lectin directly binds to target cell surface; this resembles binding of C1 to immune complexes and directly activates the classical pathway (without the need for immune complex formation).

Regulation of Complement Activity

Following factors act as a control mechanism against prolonged complement action:

i. Specific inhibitors of activation of some complement components (particularly C1 and C3) are present in plasma.

Figure 1.24: The complement pathway. Solid arrow indicates transformation of a complement component. Dashed arrow indicates enzymatic action of complement component that causes cleavage of that component

ii. Enzymatically active complement components have a very short life and are rapidly degraded to inactive forms.

iii. Active fragments are rapidly cleared from circulation.

Various Effects of Complement Activation

1. Opsonisation: Macrophages have specific receptors for C3b and thus target cells coated with C3b are recognised and phagocytosed by them (Opsonins are substances which when present on the surface of the antigen such as red cells facilitate immune phagocytosis; these are C3b and Fc portion of immunoglobulin which are recognized by specific receptors on the surface of macrophages).

2. Target cell lysis by membrane attack complex C5b-9.

3. Acute inflammation: Certain complement components play a role in acute inflammation. C3a and C5a are anaphylatoxins and increase vascular permeability.

C5a, in addition, causes neutrophil chemotaxis.

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‰ MEGAKARYOPOIESIS

The process of development of megakaryocytes and platelets in bone marrow is known as megakaryopoiesis. It is divided into four stages (Fig. 1.25). Megakaryoblasts (stage I) are the earliest morphologically recognizable precursors; they are 6 to 24 µ in diameter, contain a single, large, oval, kidney-shaped, or lobed nucleus with loose chromatin and multiple nucleoli, and have deeply basophilic agranular cytoplasm. Promegakaryocytes (stage II) are larger than megakaryoblasts (15–30 µ), have lobulated or horseshoe-shaped nucleus, more abundant and less basophilic cytoplasm which may contain azurophil granules. Granular megakaryocytes (stage III) are 40 to 60 µ in diameter, contain a large multilobed nucleus with coarsely granular chromatin, and have abundant mildly basophilic cytoplasm containing numerous azurophil granules. Mature megakaryocytes (stage IV) are of similar size, contain a tightly packed multilobed and pyknotic nucleus, and have acidophilic cytoplasm; granules are arranged as ‘platelet fields’ (groups of 10–12 azurophil granules). Sometimes neutrophils or other marrow cells are seen traversing through the cytoplasm (emperipolesis); it has no clinical significance.

Mature megakaryocytes extend long and slender cytoplasmic processes (proplatelets) between endothelial cells of sinusoids in bone marrow and platelets are released from fragmentation of these processes. Each meakaryocyte produces 1000 to 5000 platelets, leaving behind a ‘bare’ nucleus which is removed by macrophages.

A unique feature of thrombocytopoiesis is endomitosis. This refers to nuclear division with cytoplasmic maturation but without cell division. As the cell matures from megakaryoblast to the megakaryocyte, there is gradual increase in cell size, number of nuclear lobes, and red- pink granules and gradual decrease in cytoplasmic basophilia.

Megakaryocytes, the most abundant cells of the platelet series in the marrow, are large and contain numerous nuclear lobes with dense nuclear chromatin, and small aggregates of granules in the cytoplasm. The megakaryocytes possess well-developed membrane

Figure 1.25: Megakaryopoiesis

demarcation system. Upon complete maturation, megakaryocytes extend pseudopods through the walls of the marrow sinusoids and individual platelets break-off into the peripheral circulation (Fig. 1.26). There is evidence that some of the megakaryocytes are carried to the lungs where platelets are released. A humoral factor, thrombopoietin, controls the maturation of megakaryocytes.

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