Antibodies – Provide 7 Forms of Protection

Antibodies (also known as immunoglobulins, Ig) have been pivotal part of the immune systems of vertebrates, providing protection for over 500 million years!   Antibodies are glycoproteins produced by plasma B cells and can bind many types of pathogens (bacteria, viruses, fungi, and protozoa) and pathogenic toxins.  Specifically, antibodies are able to bind non-self antigens on the surfaces of pathogens and toxins and target them for destruction and recycling in order to prevent infection and damage of host cells.  Antibodies are soluble proteins that are either secreted into bodily fluids or incorporated into cell membranes to function as receptors.  The term humoral immunity refers to the “humors” (i.e. bodily fluids) being protected through the circulation of antibodies.  Antibodies bind to specific epitopes on antigens and the term antigen is derived from 2 words “antibody generator”.

In the case of non-self antigens that belong to infecting pathogen, antibodies protect their hosts through at least 7 different proposed mechanisms:

1.  Neutralization: Antibodies are able to bind to bacterial toxins and viruses and prevent them from entering and/or damaging host cells.

2.  Agglutination:  Antibodies are able to bind together cells (e.g. bacteria, foreign cells) that are displaying non-self antigens, causing them to clump together (i.e. agglutinate), reducing their ability to cause harm to the host.

3.  Precipitation:  Antibodies are able to bind together soluble non-self antigens causing these soluble antigens to precipitate and be less able to cause damage.

4.  Complement Activation:   Once antibodies are bound to non-self antigens on the surfaces of pathogens, they are able to facilitate the binding of complement proteins, which when activated in this fashion can help destroy pathogens through 3 mechanisms.  Firstly, complement proteins can create portals in the pathogen which are known as membrane attack, complexes (i.e. MAC attacks) which lead to pathogens becoming leaky.  The influx of fluid cause the pathogen to burst in a process known as immunolysis.  The second mechanism that complement proteins use for destroying pathogens is through their recruitment and activation of mast cells, which upon activation, will degranulate releasing pro-inflammatory cytokines (e.g., histamine and prostaglandins).  The inflammatory response facilitates the migration, chemotaxis, and activation of other white blood cells (WBCs) that assist in removing the pathogen either through phagocytosis or through the secretion of toxins.  Thirdly, the complement proteins act as opsonins, enhancing phagocytosis of the pathogens that they are attached to by WBCs such as neutrophils.

5.  Opsonization:  Antibodies can bind to cell surface antigens on pathogens enhancing the ability of phagocytes (e.g., neutrophils and macrophages) to bind to the pathogen, phagocytose and destroy the pathogen, in a process sometimes referred to as immunophagocytosis.  The mechanics are such that the Fv (variable antigen-binding) regions of the antibody bind to the pathogen, and the opposite side, the antibody’s exposed Fc (constant region) can then be bound by phagocytes (e.g. neutrophils and macrophages) enhancing their ability to phagocytose the pathogen.

6.  Antibody-Dependent Cell-mediated Cytotoxicity (ADCC):  ADCC begins with IgG antibodies using their Fv regions to bind to non-self antigens on virus infected cells.  This is followed by their Fc ends being bound by Fc receptors of Natural Killer cells.  Once NK cells are bound to antibodies that are attached to virus infected cells, NK cells release compounds capable of lysing the infected cell, preventing further production of viruses.

7.  Toxin Secretion Blockage:  Some antibodies have been found to block the needle-shaped tip of the toxin secretion apparatus used by some bacteria.  In this manner, antibodies that can bind to the V-antigen on bacteria in this secretion apparatus block the secretion of bacterial toxins.  This is beneficial as some species of bacteria can inject toxins directly into host cells through the secretion apparatus that is part of what is known as an injectisome.  By injecting bacterial toxins directly into host cells instead of releasing them extracellularly, antibodies and WBCs are not able to bind and destroy the toxins themselves before they cause cellular damage.  By injecting toxins, bacteria are effectively able to hide these toxins from the immune system.  In order to counter this, antibodies that target and bind the bacteria secretion apparatus, can block the secretion and injection of toxins, preventing cellular damage.

*Memory Trick:  The 7 mechanisms can be memorized through the first initial of each: NAPCOATs

Five Antibody Classes (Isotypes)

Mammals express 5 isotypes (classes) of Ig heavy chain genes (IgA, IgD, IgE, IgG, and IgM).  Interestingly even though all 5 isotypes have different shapes and functions, all antibodies consist of 4 polypeptides (2 identical heavy chains and 2 identical light chains).  Additionally, each of the 4 polypeptides have a variable domain and constant domains (Fc), with the variable domains (Fv) forming the antigen-binding region (Fab).

Let’s explore the main features of each isotype.

IgM Antibodies – Roles in Complement Activation, Adaptive Immunity, and Type II Hypersensitivity Reactions

IgM antibodies have been found to be involved in: 1) activation of the complement system, 2) playing a pivotal role in primary and secondary (specific/adaptive) immune responses, and 3) Type II Hypersensitivity Reactions.

The primordial (earliest or first) isotype of heavy chain antibodies known to exist on Earth are the IgM antibodies, first found in the jawed vertebrates dating back to the Cambrian Paleozoic Era (500 million years ago).  A diverse group of IgM antibodies are expressed by most animals today, and have many useful features.  In many species including humans, IgM antibodies able to multivalently bind specific targets (e.g., antigens, receptors) simultaneously due to their pentameric form.

IgM antibodies are produced predominantly by plasmablasts (plasma B cells) in the spleen and bone marrow, and is also produced in all lymphatic tissues.  IgM circulates the bloodstream and is widely found throughout the body.

In terms of function, IgM antibodies are able to bind complement proteins facilitating complement activation.  Furthermore, IgM antibodies are the first type of antibody to increase in concentration in the blood during a primary (adaptive/specific) immune response.  Additionally, in the case of blood type incompatibility reactions, IgM is the primary type antibody involved and is responsible for targeting the donated (i.e. foreign, non-self antigen-expressing) red blood cells for hemolysis.  These unfortunate blood transfusion errors fall into the category of Type II hypersensitivity reactions, which we will explore in more depth in subsequent sections.

IgA Antibodies – Roles in Secretions and in the Bloodstream

IgA antibodies play a crucial role in our immune system, being the most plentiful antibodies in the body, particularly within the mucosae (mucosal membranes).  IgA antibodies are responsible for protecting mucosal surfaces of the body as well as by providing passive and active protection.

IgA antibodies exist in dimer form within the mucosal surfaces of respiratory and digestive as well as skin.  Within the blood, IgA antibodies are present as monomers. and are second to IgG antibodies in terms of abundance.  IgA antibodies are most prevalent within the secretions of various bodily fluids such as tears, saliva, sweat, colostrum, mucus, and secretions of the genitourinary tract, gastrointestinal tract, and respiratory mucosa.  This broad distribution helps them defend against pathogens at mucosal surfaces and beyond.  IgA antibodies are particularly vital for infants, providing passive immunity through breast milk during breastfeeding.  However, their effectiveness can be compromised by certain bacterial species that produce proteases capable of destroying human IgA, highlighting the ongoing battle between pathogens and our immune defenses.

Although it has traditionally been thought that IgA antibodies only provide passive protection by neutralizing antigens, it has been found that IgA antibodies can also induce cytokine production resulting in stimulation of active immune responses.

IgD Antibodies – Humoral Response and Mucosal and Breast Milk Protection

IgD antibodies have the distinction of having a widespread of different functions.

Role in Humoral Immunity:  IgD has a role in B cell maturation and activation.  Although it is unclear why immature B cells express both IgM and IgD receptors that have identical antigen-binding regions, it is known that IgM receptors are down-regulated once a B cell has become activated.  It is thought that despite the redundancy of having two receptors with the same binding zone, that both IgM and IgD are necessary for:  1) B cell maturation, 2) the ability to be stimulated by both T cell dependent and T cell independent antigens, as well as 3) the development of self-tolerance.  It has been reported that the down-regulation of IgM receptors on B cells occurs during development prior to the initiation of B cell sensitization and may be important in reducing the development of an auto-immune response.  The IgD receptors that are expressed during B cell development increase and are highly concentrated in naïve B cells.  IgD antibody receptors play an important role during an immune response.  B cells become sensitized when they use IgD antibody receptors to capture and phagocytose foreign antigens, which they then display on their cell surface using MHC-II complexes.  It is thought that the form of IgD antibodies may impact its role on B cells.  IgD antibodies are longer and more flexible (bendy) than IgM antibodies.

Mucosal Surface Protection:  While IgD antibodies do circulate on their own in the blood stream, their serum levels are much lower than IgG, IgM, and IgA.  IgD antibodies are more prevalent in aerodigestive mucosal tissues and play a role in providing mucosal protection.  Specifically, IgD antibodies are found to be abundant in the upper respiratory mucosa, the tonsils as well as in the mucosa of the upper digestive tract (which includes the mouth, pharynx and esophagus).  IgD antibodies are rarely found in the spleen, and bone marrow.  It is likely the plasmablasts that produce IgD antibodies have migrated to salivary glands, lacrimal glands, mammary glands as well as the middle ear in order to secrete IgD antibodies which protect against various bacteria.  IgD antibodies, therefore, are found in saliva, tears, and breast milk.

IgD antibodies are known to bind to basophils and mast cells and induce an inflammatory response.  Interestingly IgD has been found to reduce antigen-induced IgE-mediated basophil and mast cell degranulation (and subsequent inflammation).  In fact, the higher the level of anti-allergen IgD present, the more desensitized allergen IgE mast cells become.

IgG antibodies – Role in Primary and Secondary Immune Responses and Antibody Test Results

IgG antibodies are small and are the most prevalent antibody in blood.  IgG antibodies are Y-shaped monomers that are small in size allowing for easy movement through tissues and blood vessels.  This allows for increased levels of antibody surveillence throughout the body as well as in diffusion from maternal blood vessels to the placental blood vessels and fetus providing protection in utero.  Additionally, the presence of IgGs in breast milk continues protection after the neonate is born.  Breast milk has been found to contain all 5 isotypes of antibodies, with IgA and IgG levels being the highest.

Primary and Secondary Immune Responses:  As a quick reminder, primary immune responses can be induced by vaccinations as well as by actual infections and is the reaction of immune system when it encounters a non-self antigen for the first time.  In a primary immune response, adaptive (specific) immunity is developed by T and B cells (T and B lymphocytes) that are activated mainly within the lymph nodes and spleen upon exposure to non-self antigens.  Once activated, T and B cells orchestrate targeted responses to the non-self antigens that have either been injected in the case of a vaccination, or are present on the actual infecting agent (e.g., bacteria, virus, fungus, protozoa).  This targeted response involves the proliferation of cyotoxic T cells and B cells lead to both cell-mediated and humoral (antibody) immune responses against the specific non-self antigen.  The antibodies that the B plasma cells take about 7-10 days to reach peak levels and are predominantly IgG and IgM isotypes.

Secondary immune responses are induced by exposure to the exact same non-self antigen (either through booster shot or infection) on a second or subsequent occasion   Secondary immune responses involve a very quick activation of memory B and T cells within the bone marrow, spleen and lymph nodes.  Once activated memory B and T cells proliferate rapidly giving rise to B plasma cells and cytotoxic T cells.  The B plasma cells produce antibodies that peak in 3-5 days and are predominantly IgG and IgM with some IgA, and IgE antibodies.  Both the antibodies and cytotoxic T cells target the non-self antigen for elimination.

Both IgG and IgM antibodies increase in number during both primary and secondary adaptive (specific) immune responses.  While IgM antibodies are the first to increase in number during a primary immune response, IgG concentrations rise to significantly higher levels than IgM levels in secondary immune responses and provide the most protection from the infecting agent.  

Measuring the serum levels of IgG antibodies can be helpful in determining the immune status of a individual.  For example, a person that has been vaccinated recently against measles, mumps, rubella (MMR), hepatitis B virus, varicella zoster (chickenpox), poliovirus, tetanus, diphtheria (DPT) can be tested for the presence of IgG antibodies against each of those pathogens.  The presence of serum IgG antibodies against each of these pathogens will indicate either a recent vaccination or a recent infection.  Levels can be analyzed to determine how well protected a person is against future infections.  Low levels may indicate that a booster shot is recommended, particularly in individuals that are immunocompromised.

In other cases, this type of serum IgG test can be used to determine whether a person has been previously infected with a pathogen, or is host to a chronic infection.  For  example, serum can be tested for the presence of anti-HIV IgG antibodies, and if present, indicate that the individual has been infected with HIV.

IgE antibodies – Defends against Parasitic Worms (Helminths) and Protozoa and has a role in Type I Hypersensitivity Reactions

IgE is the least abundant isotype of antibody present in the body and in the blood, though is important in providing defense against parasitic worms (helminths) and protozoa.  Unfortunately, IgE also plays a pivotal role in the development of Type I hypersensitivity reactions which include allergies as well as asthma.  IgE binds to Fc receptors on the surface of basophils and mast cells and stimulates these cells to degranulate releasing pro-inflammatory cytokeins (e.g., histamine, prostaglandins, and leukotrienes).  This activation of degranulation occurs when the IgE binds to an antigen which is helpful when the antigen belongs to a pathogen, as inducing inflammation and activation of WBCs and the immune response can lead to the elimination of the pathogen preventing further damage to host cells.

Interestingly, IgE antibodies are produced by plasma B cells, and free IgE have a short half-life in the bloodstream, however, due to the high affinity receptors on basophils and mast cells, IgE quickly become bound to these cells, which is thought to increase their half-life significantly.  It is thought that this can be beneficial in protecting against pathogens as well as venoms (including bee sting venom).   It has been found that by becoming sensitized to small amounts of bee sting and viper venom can help provide resistance against future larger and potentially lethal doses of venom.

However a condition termed atopy can occur, and this refers to the over-production and persistence of IgE and persistence in response to a harmless substance (e.g., an allergen such as pollen), leading to the development of allergies and allergic symptoms.  In the unfortuanate case of bee sting allergies, likewise, the exaggerated IgE immune response and over-activation of mast cells and basophils in a Type I Hypersensitivity reaction leads to the allergic signs and symptoms.

*Memory Trick:  The 5 isotypes of antibodies can be remember through the acronym MADGE.

Summary:

• Antibodies (Immunoglobulins, Ig):
  • Glycoproteins produced by plasma B cells comprised of 2 identical heavy chain polypeptides and 2 identical light chain polypeptides with Fv, Fab and Fc regions.
  • Fc constant region that can bind to Fc receptors on WBCs (e.g., neutrophils) to assist with phagocytosis
  • Provide humoral immunity by binding various non-self antigens on infecting pathogens (bacteria, viruses, fungi, protozo, helminths) and toxins.
  • Facilitate destruction of pathogens

• Mechanisms of action (NAPCOAT)
  • Neutralization: Prevent pathogens from entering or damaging host cells.
  • Agglutination: Clump together pathogens to reduce harm.
  • Precipitation: Cause soluble antigens to precipitate, reducing harm.
  • Complement Activation: Assist in pathogen destruction via MAC attack, inflammation, and opsonization.
  • Opsonization: Enhance phagocytosis of pathogens by immune cells.
  • Antibody-Dependent Cell-mediated Cytotoxicity (ADCC): Induce target cell lysis by NK cells.
  • Toxin Secretion Blockage: Block bacterial toxin secretion to prevent host cell damage.

• Five Antibody Classes (Isotopes):
  • IgM:
    • Large pentameric form, prevalent in blood and lymphatic tissues
    • Activates complement system,
    • First to increase in primary immune response
    • Involved in blood type incompatibility reactions (Type II Hypersensitivity Reactions).
    • Attacks wrong blood cells in transfusion errors.
  • IgA:
    • Most prevalent antibody in the whole body, abundant in mucosae
    • Secreted outside the body (tears, saliva, sweat, colostrum, breast milk, mucus, and secretions of the genitoruinary tract, gastrointestinal tract, and respiratory mucosae).  Also found in blood stream
    • Provides passive protection, can also activate immune response
    • Some bacteria species release proteases that destroy human IgAs.
  • IgD:
    • B cell receptor, Role in B cell maturation, sensitization and activation
    • Also found in mucosal tissues and breast milk
    • Involved in down-regulating allergies 
  • IgG:
    • Small, most common antibody in blood
    • Involved in primary and secondary immune responses
    • IgGs are the predominant antibody produced during subsequent infections (secondary immune responses)
    • Provides widespread immunity, passes through placental barrier to fetus, found in breast milk and mucosae
  • IgE:
    • Defends against protozoa, helminths (parasitic worms) and venom
    • Associated with mast cells and basophils.
    • Involved in Type I Hypersensitivity reactions (allergies and extrinsic asthma)
    • Can cause allergies by triggering inflammation through histamine release.

• • Applications:

• • • Serum IgG levels used to assess immune status post-vaccination or infection.
    • Some bacteria release proteases that destroy antibodies.
    • Some cancerous cells are recognized by antibodies.
    • Antibody therapies are being researched and developed to fight cancer.