Humoral immunity

Introduction:

Humoral immunity is the type of host defence that is mediated by antibodies, the products of B cells. Antibodies are secreted into mucosal lumens, the blood, and interstitial fluids, and combat microbes at all these sites. As already stated B-cells are the source of antibodies, let us first understand the development and maturation of B-cells. During embryogenesis B cells are first recognized in the liver. From there they migrate to bone marrow they don’t require thymus.

Phases of B-Cell development and maturation:

Stages of B-Cell development: Stem cells, Pre-B Cell, B-cell

  • 1ST PHASE is s independent phase which consist of stem cells, pre B-cells & B-cells.
  • 2nd PHASE is antigen dependent phase which comes into play due to interaction of antigen with B-cell. Ig-M is displayed on B-cell surface first which act as receptor for antigens (sometimes IgD).

B cells constitute about 30% of circulating lymphocytes. Life span ranges from days to weeks. Approximately 109 B-cells are produced each day. There is a large pool of B –lymphocytes approximately 107 with different specificities.

Hapten: Foreign material that is recognized by the immune system, but can only function as an immunogen when complexed to other immunogens.

Activation of B-Cells:

Multivalent antigens directly attach to IgM and IgD on B cell surface. It is due to cross linkage of antigen with antibodies on B-cell surface. Another way is via T-cells, through secretion of IL-4 and IL-5. Important is co-stimulatory interaction between CD-40, CD-40L and B7, CD28.

Antibody Structure and Humoral Response:

Soluble proteins that circulate in body and perform major functions of immune system are antibodies. Initially when found they were called as ϒ globulin (migration on field) but today they are called Immunoglobulins. Two most important features of Immunoglobulins are specificity and biological activity. These can identify large numbers of antigens. One part of antibody is adaptable to large number of epitopes and the other part is adapted to participate in biological activities.

Isolation of Immunoglobulins:

Electrophoresis of serum gives five major components: Albumin, α1, α2, β and ϒ globulins. In these, ϒ globulin is least migrating. It forms a very small peak so were difficult to identify but in case of multiple myeloma, immunoglobulins are elevated called as Bence Jones proteins (In 1845, Henry Bence Jones discovered immunoglobulin proteins in urine of multiple myeloma patients), so this peak is particularly high.

Structures of Light and Heavy Chains:

Porter in 1962 found that proteolytic treatment of enzymes papain splits Immunoglobulins in to three parts of almost of equal molecular weight. Two of these parts can bind to antigen but no longer precipitate. These two parts are called as fragment antigen binding (Fab). Third fragment could be crystallized due its property indicative of homogenicity so it was called as fragment crystallizable (Fc). It could not bind to antigen but it could participate in biological functions. His data lead to the proposal of a Y-shaped structure of antibody. At the same time Edelman in US discovered that ϒ globulins were extensively reduced by treatment with mercaptoethanol (agent that breaks disulfide bonds). It broke Immunoglobulin molecule into four chains, two heavy chains (H chains) of identical molecular weight upto 53,000 daltons and two light chains (L chains) having molecular weight 22,000 daltons. On the basis of these findings they proposed structure of Immunoglobulins and got noble prize.

Basic structure of Immunoglobulins:

  1. Immunoglobulins of one species are antigenic to other species.
  2. Light chain contains about 211 to 217 amino acids amino acids and heavy chains contain approximately 450 amino acids.
  3. By studies it has been shown that all the species have only two types of light chains λ and κ. Every individual in a species synthesizes both of these chains but ratio i.e. λ/κ varies from species to species (mouse 95% κ and human 60% of κ). But in any immunoglobulin molecule both light chains are same that is either λ or κ.
  4. Heavy chains of virtually all species consist of five different classes (isotypes) that differs in their structure of heavy chains. H chains of different Immunoglobulins are IgM-µ, IgG-γ, IgA-α, IgE-ϵ, IgD-δ. Two heavy chains in a Immunoglobulins molecule are same e.g. IgG molecule can have structure κ2γ2 or λ2γ2.
  5. H chains confers on a molecule its unique property such as half life in circulation its ability to bind certain receptors and activate enzymes.
  6. Further subdivision of these molecules gives subclasses for example IgG has been divided into IgG1, IgG2, IgG3, IgG4. In the same way IgA has been divided into IgA1 and IgA2 .These sub isotypes vary in number and arrangement of interchain disulfide bonds.
  7. L and H chains are subdivided into variable and constant regions. These regions are composed of 3-D folds called as ‘domains’. These disulfide bonds are present within a chain and between two chains. In a chain, these disulfide bonds make loop called as domains. Most of H chains contain one variable that is (VH) and three constant (CH) domains. IgG and IgA have three CH domains where as IgM and IgE have four CH domains. Each domain contains about 110 amino acids.
  8. VH and VL are responsible for antigen binding whereas constant region is responsible various biological functions such as complement fixation and binding to cell surface receptors. Certain amino acids in hypervariable regions are very variable. Only 5 to 10 amino acids in each hypervariable region from binding site. There are three hypervariable regions present in both VH and VL. If we count from amino acid terminal these are found in and around 30, 50 and 95 amino acid regions which are called as complementary determining regions (CDR’S). Antigen-antibody bonding involves electrostatic and vander walls forces. In humans, immunoglobulin containing κ chains to those containing λ chains is approximately 2:1.

Antibody structure

Antibody structure

Individual Immunoglobulin:

Immunoglobulin G:

Predominant Immunoglobulin in blood that makes about 75% of total immunoglobulins. Molecular formula is H2L2. It is a monomer and is divalent (has two identical antigen binding sites). It has got four subclasses with a ratio IgG1 : IgG2 : IgG3 : IgG4 = 66 : 23 : 7 : 4. It is synthesized in fetus as early as 20 week of gestation. Molecular weight is about 150000 D. sedimentation coefficient 7S. Electrophoretically IgG molecule is least anodic and migrate to gamma range. Except for variable region all immunoglobulins in one class have 90% homology. So antisera are against all classes 1-4. Half life of IgG-1 is 23 days. It is same for IgG1, IgG2 and IgG4. IgG3 has half life of 7days. IgG-1 is responsible for erythroblastosis fetalis.

Functions:

  1. Opsonization.
  2. Antibody dependent cell mediated cytotoxicity (ADCC): Virus infected cells can be destroyed by combination of IgG and phagocytic cells. Antibody binds to the surface of the infected cell. This antibody is recognized by receptor for this antibody present on phagocytic cell i.e. macrophage or NK cell. The infected cell is killed. This is main defense against helminthes infections but in this case IgE is involved and eosinophils are effector cells.
  3. Activation of complement.
  4. Neutralization of toxins.
  5. Immobilization of bacteria by clumping their flagella and cilia.
  6. Neutralization of viruses (by attaching to surface of virus and preventing its attachment to target cell)

Immunoglobulin M:

IgM is the first immunoglobulin to be produced after immunization. It is found in monomer or pentamer structure. For pentameric structure the mol. weight is 900000 daltons and has sedimentation coefficient of 19s. 5 units are joined to each other by disulfide bonds. A polypeptide chain joins the Fc portion of monomer units called as J chain. This J chain is synthesized in B-cell or plasma cells like Antibodies.  It has wt. of 15000 daltons. Valancy of IgM is 5 instead of 10 because due to congested structure only 5 antigen epitopes can attach as the Fab portion can’t open fully. If IgM is found to be raised in newborn, it is indicative of intrauterine infections of rubella, syphilis, toxoplasmosis, CMV infections. It is an active activator of compliment system by classical pathway.

Immunoglobulin A:  

It is the major immunoglobulin present in external secretions such as saliva, mucous, sweat, gastric fluid and milk. It provides neonate with protection against intestinal infections. Mol. Wt. is around 165000 daltons. Its sedimentation coefficient is 7s migrates to β or fast γ region .It may exist as monomer or dimer. In secretions it is secreted as dimer. These are united together by a J chain. These units are covalently joined by J chain. It also has another polypeptide chain called as secretory component. Joining chain is a product of plasma cells. Secretory component is the product of epithelial cells and plays an active role in transportation of IgA into various secretions.

Table describing various properties of antibodies

Type of Immunoglobulin

Heavy chain

Light  chain

MW(kDa)

Structure

Function

IgA1  IgA2

α1
α2

λ or κ

150 to 600

Monomer to tetramer

Mucosal immunity: secretion of IgA into lumens of gastro-intestinal, respiratory tract etc.

IgD

δ

λ or κ

150

Monomer

Antigen receptor for naive B-lymphocytes.

IgE

ε

λ or κ

190

Monomer

Antibody dependent cell mediated cytotoxicity involving eosinophils.
Mast cell degranulation (immediate hypersensitivity reactions).

IgG1 
IgG2a
IgG2b
IgG3 
IgG4

γ1,γ2,γ3,γ4

λ or κ

150

Monomer

Opsonization of antigens for phagocytosis by macrophages and neutrophils.
Activation of the classical pathway of complement.
Antibody-dependent cell mediated cytotoxicity mediated by natural killer cells and macrophages.
Neonatal immunity: transfer of maternal antibody across the placenta and gut.
Feedback inhibition of B cell activation.

IgM

µ

λ or κ

900

Pentamer

Activation of the classical pathway of complement.
Antigen receptor of naive B lymphocytes.

Immunoglobulin D:  

IgD is present in serum at very low concentration and amount varies. It is probabesly not secreted by plasma cells. It is not designated any definite function. But it is said to be associated with IgM as surface component of many B cells. It is monomer with mol. wt. around 150000 dalton and sedimentation coefficient of 7S.

Immunoglobulin E:

It has extra CH domain. Monomer with mol. wt. around 190000 D. Sedimentation coefficient is 8S. Migrates to fast γ globulin region. IgE is called as “reaginic- antibody”. It is present in serum at lowest concentration. It has specific domain on its H chain which attaches with the mast cells with high affinity. It attaches to mast cells and basophils through its receptors spaning between FcR1 -> CE2 -> CE3 junction in Fc region. There are many cell surface receptors for FcR1 (antibodiesout 104 on basophils and  106 on mast cells). When two IgE molecules are cross linked by on cell it becomes active and secretes its contents.

Some facts about antibodies:

  • Highest concentration is that of IgG.
  • Fetal synthesis of IgM and IgA begin during the 5th month.
  • Plasma cells can secrete IgM, IgG, IgA, IgE (all but IgD).
  • IgA has 2 protions: J chain and S component.
  • IgM also has J chain.
  • Mg/ml = IgG: IgA : IgM : IgD : IgE = 12 : 1.8 : 1 : 0-0.04 : 0.00002
  • Immunoglobulin present on lymphocyte surface is IgD and on basophils and mast cells is IgE.
  • Half lives:

                                                 IgG – 23 Days

                                                 IgA – 5.5 Days

                                                 IgM- 5 Days

                                                 IgD – 2.8 Days

                                                 IgE – 2 Days

  • Placental passage  is only for IgG
  • Only IgA is present in secretions.
  • IgG and IgA are present in milk.
  • Complement activation is mainly by IgM but also by IgG.
  • Antiviral activity is highest with IgGs and IgA (IgM also)
  • Antibacterial activity is by IgG, IgA, IgM.
  • Antitoxin activity is only with IgG.
  • Allergic activity is with IgE.

Light and heavy chains of human antibidies

Immunoglobulins light and heavy chains

Isotypes:

Isotypes of antibodies are formed due to the difference in the amino acid sequence in their constant region. For example IgG and IgA are isotypes as their heavy chains are different antigenically. Individual immunoglobulin can have its subtypes such as IgG has its subtypes IgG1, IgG2, IgG3, and IgG4 (based on antigenic difference on their heavy chains).

Allotypes:

Some features of antibodies vary from person to person for example IgG has 2H and 2L chains. Genes which code for γ chain are polymorphic. Every individual is inherited different alleles which changes the amino acid sequence from person to person.

Idiotypes:

These are antigenic determinants formed by specific amino acids present in hypervariable region. Each idiotype is unique for an antibody producing cell.

Summary:

  • Constant region on heavy chain determines the immunoglobulin class.
  • Constant region on heavy and light chains determine the allotypes.
  • Variable region of light and heavy chains determine idiotypes.
  • Constant region of heavy chains bind IgG to macrophages.
  • Fixation of complement is also in constant region of heavy chains.
  • Variable regions of light and heavy chains are antigen binding sites.
Know more…………

Immunodeficiency: There are 4 main components of immune system:

1)      B-cells.

2)      T-cells.

3)      Complement.

4)      Phagocytes.

The Immunodeficiency diseases can be classified as congential or Acquired. Recurrent or opportunistic infections indicate immune deficiency. Opportunistic infection with pyogenic bacteria indicates B-cell deficiency where as opportunistic infections with fungi, viruses and protozon indicate T-cell deficiency.

I)     Congenital B-Cell Immunodeficiency:

  • Burton’s agamaglobunimia: This is X- linked disease so only males are affected.  In this condition all Immunoglobulins are decreased two very levels and deficiency of B-cell is also there. Pre-B cells are present but they don’t form mature B-cell. This is due to lack of enzyme Tyrosin Kinase. Cell mediated immunity is normal. Patient starts having pyogenic infections after 6 months of birth as maternal IgG protection is reduced at this time.
  • Selective Immunoglobulin deficiency: Most commonly IgA is deficient. IgG & IgM deficiencies are very rare. Patient with IgA deficiency has recurrent sinus & lung infection.

II)   Congenital T-Cell deficiency:

  • De’george Syndrome (Thymic Aplasia): This disease is due to defect in third & fourth pharyngeal arch due to which thymus & parathyroid fail to develop. Early in life patient suffer from severe viral, fungal or protozoal infections due to profound deficit in T-cell. Most common symptom is tetany due to hypocalcemia. Thymus transplant from a child less than 14 years cures a patient. Chronic mucocutaneous candidiasis occurs in severe T-cell deficiency.
  • Hyper IgM Syndrome: Patient has high level of IgM but low level of IgG, IgA, and IgE etc. T & B cells are normal.
  • Combined B Cell & T-Cell Deficiencies (SCID) Severe Combined Immunodeficiency Disease: Recurrent infections caused by fungi, bacteria, viruses, protozoans occur in early pregnancy because both B-cell & T-cell are defective. It is inherited as two types; X-linked & autosomal. Patients who have mutant tyrosin kinase gene ZAP-70 & adenosine deaminase & purine nucleoside P. phosphorylase can suffer from B-cells & T-cells deficiency.
  • Wiskott-Aldrich Syndrome: X-linked disease that occurs in male infants. Patient has inability to mount B cell response. Basically T-cell response is defective which is not able to provide help to B-cells proliferation.
  • Ataxia-Telegiectasia: Ataxia (staggering), telengiectasia (malformed in larger blood vessels)

 

References:

  1. Immunology: Understanding The Immune System. By Klaus D. Elgert
  2. Immunology: A Short Course. By Richard Coico, Geoffrey Sunshine.
  3. Immunology.  By David K. Male.
  4. Immunology: Essential And Fundamental. By Sulabha Pathak, Urmi Palan.

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