Central Lymphoid Organs

The following points highlight the ten major types of central lymphoid organs. The types are: 1. Thymus 2. Bone Marrow 3. Lymph Nodes 4. Spleen 5. Mucosa Associated Lymphoid Tissue (MALT) 6. Cells of Lymphoreticular System 7. Delayed Type Hypersensitivity T (Tdh Cells) 8. B-Lymphocytes and Plasma Cells 9. Microphages 10. Major Histocompatibility (Complex) (MHC).

Thymus:

Thymus is a lymphoepitheial structure situated behind the upper part of sternum. It consists of two lobes. Each lobe contains several lobules. Each lobule is made of an outer cortex crowded with small lymphocytes and an inner medulla consisting of epithelial cells (thymocytes).

The thymus develop into area of lymphoid cells and epithelial cells forming cortex and medulla, respectively. Maturation of lymphoid cells in thymus is most vigorous during foetal and neonatal development and proceeds till puberty.

Production of thymic lymphocytes is the primary function of thymus. During maturation process, the surface membrane of lymphocyte acquires new antigen (Thy antigen) from thymus and these thymus processed cells are called T-lymphocytes (thymus dependent).

These immunologically competent T-cells, after release from thymus, circulate through blood and lymphatic’s and constitute 75% of the circulating lymphocytes in man. The life span of the T-cells is much longer than B-cells (i.e. months or years).

Bone Marrow:

Within the bone marrow some lymphoid stem cells develop into immunoglobulin (Ig) producing lymphocytes known as B-cells, because they mature into bone marrow. In chickens and birds, B-lymphocytes mature in the (lymphoepithelial organ). The bursal equivalent in mammals is bone marrow. It is the site for both stem cells proliferation and origin and maturation of pre B-cells into functionally mature B-lymphocytes.

The lymphoidal stem cells (pre B-cells) lack surface immunoglobulin but display intracytoplasmic mµ (µ) chains but no light chain. B-cells express IgM on their surface in early stage of their differentiation as the principal antigen receptor. They lack both C₃ and F_c receptors at this stage.

At the next stage of differentiation B-cells bear either surface IgM alone or in combination with IgA or IgG depending upon the commitment to the production of a particular antibody class. Then IgD is also added to the surface of B-cells in addition to C₃ and F_c receptors required for the entrance of B-cells into mature phase.

The cells now acquire receptors for immunoregulatory factors. These cells are called virgin cells as they have not yet been exposed to any antigen. Thus some virgin cells contain surface immunoglobulin of three different classes Ig M, IgG and IgD or IgM, IgA and IgD but all the immunoglobulin molecules on a single cell have the same idiotype.

Lymph Nodes:

They are bean-shaped and arranged along the course of lymphatic vessels. They are surrounded by a capsule from which trabeculae penetrate into the nodes.

A mature lymph node has:

(a) Outer cortex

(b) Inner medulla, and

(c) Para cortical zone between the cortical follicles and base of medullary cords.

Circulating lymphocytes of blood enter lymph node by penetration through specialised vessels. The cortex contains lymphoid follicles which may be primary and/or secondary.

Primary follicles consist of an accumulation of small lymphocytes.

Secondary follicles develop during antigenic stimulation. They are large and possess germinal centres. Medullary cords are finger like processes formed by lymphocytes.

The T and B-lymphocytes are largely separated into different anatomical components of lymph node:

i. B-cell areas:

The cortical follicles contain B-cells.

ii. T-cell areas:

The Para cortical area contains T-lymphocytes and constitutes thymus- dependent area.

Both T and B-lymphocytes migrate to sinusoidal spaces of medulla and pass into blood via different lymphatic vessels.

B lymphocytes are scattered in Para cortical area, T lymphocytes are present in and around the germinal follicle. Active cooperation between the two types of lymphocytes and the antigen presenting cells in the production of an immunological response is indicated by the distribution of T and B-cells.

The lymph node acts as filter for lymph draining the body tissues and foreign antigens which enter the sub capsular sinus by afferent lymphatic vessel. The dendrite macrophages capture and process the antigen. This helps the proliferation and circulation of T and B cells. The lymph passes via afferent lymphatic’s.

Spleen:

It is the lympho-vascular organ. It is without lymphatic supply. It processes material directly from the blood stream and it is divided into compartments by trabeculae extending from the capsule.

It has two areas—white and red pulp.

It has also:

(a) Cortex made up of densely packed B and T lymphocytes

(b) Loosely structured medulla. Unlike lymph node, medulla is external to cortex.

Spleen is an important organ of antibody production due to presence of large number of B cells.

Like lymph node, T and B cells areas are segregated.

B-cell area:

Perifollicular region, germinal centre and marginal zone.

T-cell area:

Thymus dependent area includes the lymphatic sheath and is adjacent to the central arterioles.

Mucosa Associated Lymphoid Tissue (MALT):

The sub epithelial accumulations of lymphoid tissue guard immunologically the mucosa of alimentary, respiratory and genito-urinary tracts which are constantly exposed to numerous antigens.

The lymphoid tissue may be present as diffuse collection of lymphocytes, plasma cells and macrophages throughout the lung and lamina propria of the alimentary tract or as specialised aggregates with well-formed follicles (lingual, palatine, pharyngeal, tonsils, small intestinal peyer’s patches and appendix.

These are collectively called mucosa associated lymphoid tissues. MALT contains both B-cells and T-cells and phagocytes. It forms a separate inter­connected secondary system consisting of Ig A or Ig E producing cells.

In the alimentary canal, the antigen passing through the specialised epithelial cells enters Peyer’s patches and stimulate the lymphocytes. These stimulated lymphocytes are drained into the lymph which ultimately reach the blood stream via thoracic duct. They pass into the lamina propria from the blood stream where they become Ig A forming cells, B-cells predominate in gut associated lymphoid tissue.

Lymphocytes in Peripheral Blood:

In the circulating blood, about 75% lymphocytes are T-cells, 20% B-cells and 10% killer (K) cells.

Cells of Lymphoreticular System:

Lymphocytes:

They are small round cells in blood, lymphoid organs and other tissues. They may be small (5-8 µm), medium (8-12 µm) and large (12-15 µm). They have a thin rim of cytoplasm and spherical nucleus. The small lymphocytes are most numerous and devoid of endoplasmic reticulum. The life-span of short-lived lymphocytes is 10-20 days and that of long-lived lymphocyte is 100-200 days.

In the peripheral blood of an adult, lymphocytes constitute 25-45% of leucocytes, in lymph and lymphoid organs, they are predominant. Lymphocytes educated by central lymphoid organ become Immunologically competent cells (ICCS). Majority of long lived small lymphocytes are thymus derived.

The short lived ones serve as effector cells in immune response, while long lived ones are store houses of immunological memory. Nucleus of lymphocyte is indented B-cell and similar to T-cell with more cytoplasm and rough surface endoplasmic reticulum.

Lymphocyte traffic is the process of lymphocyte circulation after lymphopoiesis in the central lymphoid organ and peripheral lymphoid tissues. When an antigen encounters B-cells in spleen and on lymph nodes, B-cells are activated and transformed into plasma cells which synthesise antibodies.

When T-cells encounter specific antigen, they produce certain activation products (lymphokine) and induce Cell Mediated Immunity (CMI).

Laboratory Tests for Detection of T and B-cells:

(i) Sheep erythrocytes surface receptors bind around the T-cells forming rosette.

(ii) B-cells rosette is developed by binding of IgG coated sheep erythrocytes in presence of C₃ forming a complex.

(iii) B-cells have immunoglobulin’s on surface and surface receptors for Fc fragment of IgG which are absent on T-cells.

T-lymphocytes:

Thymus derived lymphocytes (T-lymphocytes) have characteristic membrane bound markers or receptors which they acquire in the thymus during maturation from lymphoid stem cells. T-lymphocytes react only with antigen on the surface of another cell, whereas B-cells react with antigen either in solution or on a cell surface.

Important T-cell receptors are:

(a) Enzyme receptor,

(b) La protein receptor,

(c) T-cell antigen receptor, CD₄.

T-cells serve as receptor for Immunodeficiency virus (HIV).

Differentiation of T-cells:

Thymic lymphocytes acquire thymic antigens or markers (detectable by monoclonal antibodies) which were previously designed by the letters T₁-T₁₁ These markers or surface molecules of T-cells are now designed as CD (clusters of differentiation) proteins.

As T-cells migrate from cortex to medulla of thymus, they undergo a series of differentiation. As they mature they begin to express their characteristic cell membrane glycoprotein (gp).

The T-cell receptor is responsible for the recognition complexes and is different for every T-cell. So far more than fifty T-cell antigens are identified by monoclonal antibodies.

A brief outline of the relevant CD molecules is:

CD₁:

It is a cortical thymocyte marker associated with beta globulin. It is found in early stage of maturation, but disappears at later stage.

CD₂ (T₁₁):

It is an early antigen and acts as a receptor for sheep red blood cells. It is involved in cell adherence and T-cell activation.

CD₃ (T₃):

It is CD₃ antigen is present in all T-cells and has a constant structure and associated with T-cells receptor cell membrane.

CD₄ (T₄):

It is present on 65% of the circulating T-lymphocytes and indicates helper function. It is also found on macrophages and monocytes. CD₄ recognises antigen by major histocompatibility complex (MHC) class II molecule.

Functional subset of T-cells:

T helper (CD₄ cells). It has two subsets—Th₁ Th₂. Th₂ is the principal helper T-cells which interacts with B-lymphocytes and promote proliferation of resting B-cells and development of plasma cells that produce immunoglobulin.

Delayed Type Hypersensitivity T (Tdh Cells):

T-cells are also responsible for delayed type of hypersensitivity. It is not possible to distinguish Th cells from Tdh cells on the basis of surface markers. It is believed that Tdh cells are one type of helper cell (Th₁). They bear CD₄ markers.

Th₁ cells secrete different lymphokines (eg. gamma interferon) which are responsible for inflammatory response of delayed hypersensitivity. Growth factors secreted by Th₁ are also believed to regulate lymphocyte activity.

T suppression (CD₈) cells:

Some CD₈ cells block immune response of B-cells by secreting suppressor substances which are called T suppressor (Ts) cells. They block immune response by their action on Th or B-cells.

Cytotoxic (Tc) cells and CD₈ cells:

It lyse specific target cells whose surface antigens can be recognised by them, eg. virus infected host cells, tumour cells and allogenic graft cells in transplanted tissues. Cytotoxic T-cells possess membrane glucoprotein (CD₈). Ts cells can be differentiated from Tc cell in vitro.

Natural killer (NK) cells:

Large lymphocytes containing azurophile granules in the cytoplasm are called large granule lymphocytes (LGL). NK cells are spontaneously cytotoxic for certain tumour lines, virally (herpes, mumps) transformed target cells and are involved in allograph rejection. They do not require antibody for activity and their action is now specific.

They can recognise altered surface structure of target cells. Interferon and other agents that activate macrophages (like BCG vaccine) enhance the activity of NK cells. They are found in spleen and peripheral blood of men and animals. It is believed that NK cells play an important role in controlling the development of neoplastic cells (anti-tumour immunity) and viral replication (anti-viral immunity) in the body.

B-Lymphocytes and Plasma Cells:

The pre B-cell is programmed to produce only one class or subclass of immunoglobulin after a switchover from initial IgM production which occurs as a result of further series of gene rearrangement. B-lymphocytes can be subdivided into nine different isotypes or subsets—on the basis of Ig they are programmed to synthesise IgM, IgD, IgG₁, IgG₂, IgG₃, IgG₄, IgA, Ig A₂, IgE.

The virgin B cells migrate to spleen and lymphoid tissues where a cognate Ag can trigger the second or Ag dependent stage. Majority of resting tissues and blood B-lymphocytes are small with condensed chromatin and have IgM and only a small proportion (10%) of B cells have IgG on their surface.

B-lymphocytes with IgA or IgE are abundant in association with exocrine gland of intestine, lungs where T helper cells also predominate. Each B lymphocyte carries about 10⁵ antibody molecules on its surface.

B-cells in peripheral lymphoid organs are quiescent on resting cells and the immunoglobulin is an integral membrane protein. In eliciting a primary immune response. The antigen encounters innumerable lymphocytes all bearing different antibodies and recognition site.

The antigen only binds to those Ig molecules on the surface of B-cells which it matches and can be recognised by the cell. The antigen recognition binding site is in hyper variable region of both heavy and light chains of Ig.

Macrophage presents the processed antigen to B-cells. The antigen binds to the receptor of B-cell. B-cell is stimulated to undergo blastoid transformation which divides rapidly becoming successfully plasma cell, blasts (clone formation), intermediate transitional cells and plasma cell.

Mature plasma secretes antibody. In plasma cell, the nucleus is eccentric and contains coarse radially arranged chromatin (cart-wheel appearance). The cytoplasm contains conspicuous Golgi apparatus and abundant endoplasmic reticulum. Immunoglobulin remains localised in the endoplasmic reticulum where it sometimes forms aggregates called Russell bodies.

A plasma cell can only synthesise an antibody of a single specificity either IgM or IgG or Ig A. An exception is found in primary immune response, when a plasma cell producing IgM, initially, may later switch over to the synthesis of IgG antibody. Mature plasma cells are end cells and survive only a few weeks and die after a few (or no) cell division.

Plasma cells are present in germinal centres of lymph nodes, spleen and diffuse lymphoid tissues of alimentary and respiratory tracts. Although plasma cell is the major antibody producing cell, lymphocytes, lymphoblast’s and transitional cells may also produce antibody.

Macrophages may be:

(1) Free or wandering macrophages in tissues and monocytes in the blood stream;

(2) Fixed (literal) or stationary or resident macrophages.

Monocytes originate from mono-blasts in the bone marrow, travel as monocytes in the blood stream and emigrate in tissues within two days where they enlarge and differentiate into macrophages and may live for months.

In tissues, these mononuclear cells are distributed as free macrophages (in lung alveoli; peritoneum and inflammatory granuloma) or fixed macrophages integrated in tissues (cells lining sinusoids of liver, spleen and lymph nodes), central nervous system (microglia) and connective tissues (histiocytes).

Blood macrophages (monocytes) and tissue macrophages. They may proliferate locally under certain inflammatory stimuli. Multi-nucleated giant cells and epitheloid cells in certain inflammatory processes originate from macrophages.

Monokines:

Monocytes and macrophages produce a number of important soluble proteins called monokines (cytokines).

These monokines include interleukin 1, various colony stimulating factors—such as macrophage colony stimulating factor, M-CSF; granulocyte colony stimulating factor, G-CSF; and tumour necrosis factor (TNF):

i. Interleukin 1:

(IL-1) human interleukin-1 consists of two proteins IL-1a, IL-lb, produced by macrophages—it is mediator of inflammation, causes release of acute phage of proteins.

ii. Colony stimulating factor (CSE):

It is produced by macrophages and stimulates division and differentiation of stem cell. Granulocyte colony stimulating factor promotes granulocyte differentiation process by binding to stem cell receptor. Granulocyte monocyte colony stimulating factor (GM-CSF) stimulate both granulocytes and monocyte cell differentiation.

iii. Tumour necrosing factor (TNF):

It synthesised by macrophage is responsible for the tumoricidal activity of macrophages.

Function of macrophages:

A. Active phagocytosis.

B. Role in acquired immune response.

A. Active Phagocytosis:

Engulfment of particles by macrophages is phagocytosis; engulfment of soluble material by phagocytes is pinocytosis. The membrane of the pseudopod of macrophage fuses to form a pouch or phagosome. Lysosome containing hydrolytic enzyme migrates towards phagosome and fuses with the membrane of phagosome forming phagolysosome.

Microorganisms or particles sensitized by antibody are more readily phagocytosed. Most bacteria are killed intracellularly within few minutes by lysosomal enzyme, some salmonella, Brucella, Mycobacterium resist digestion and may grow intracellularly to other sites for transportation to other sites.

B. Role in Acquired Immune Response:

Macrophages play an important role in immune response. They trap and process bacterial antigens and present them in optimal concentration to the lymphocytes to induce specific immune response. It is a prerequisite for some antigens.

The activation of resting T-cells require Interleukin-1 (IL-1) and Interleukin-2 (IL-2). Activated macrophages secrete IL-1 which acts as endogenous pyrogenes and induces to T-cells to synthesise IL-2. IL-2 facilitates T-cell activation by rapid DNA synthesis, cell mitosis and T- cell clone formation.

Microphages:

Microphages (granulocytes) include polymorphonuclear leucocytes (neutrophils, eosinophil’s, basophils).

Neutrophils:

It plays an important phagocytic role in acute inflammation by locating a foreign invader. Migrating towards it and finally engulfing the offending agent lysosome in their granules are discharged into the phagocytic vacuoles which hydrolyse red cells, many bacteria of low virulence.

Eosinophils:

These are less pathogenic than neutrophils and their granular contents (various hydrolytic enzymes) can kill parasites which are too large to be phagocytosed by neutrophils. Their numbers are more in certain allergic inflammation, parasite infections and diseases with formation of antigen-antibody complex. Functions of eosinophil’s is not yet clear.

Basophils:

These are found in very small numbers (0.2%) in blood and tissues (most cells). Basophilic granules contain certain heparin, histamine, serotonin and other hydrolytic enzymes. They possess plasma membrane receptors for the F_c portion of IgE and cross-linking of this immunoglobulin by antigen leads to release of pharmacological mediations in anaphylaxis and atopic allergy.

Immune Phagocytosis:

There are many antigens particularly capsulated bacteria which resist phagocytosis and for dealing with these organisms successfully phagocytic cells require to utilise the process of immune phagocytosis. Mononuclear phagocytes, neutrophils and eosinophil’s have receptors for F_c portion of IgG and some activated components of C₃1 and C₃ li.

During the course of an immunological response the ability of neutrophils to phagocytosis is enhanced by opsonisation. Possibly, most of the pathogenic bacteria are disposed by opsonic phagocytosis. Once the bacteria are killed by lysosomal enzyme, their degradation products are released to the exterior.

Myco. tuberculosis, Listeria monocytogenes, Leishmania sp, Trypanosoma cruzi, Toxoplasma gondii:

They can survive inside the macrophages and are able to evade the microbicidal system.

Major Histocompatibility (Complex) (MHC):

Transplants from one individual to another genetically non-identical individual of the same species are recognised as foreign by T-cells and rejected. The responsible antigens on the grafted cells are called Transplantation or Histocompatibility (or H) antigens.

The major histocompatibility complex (MHC) antigens are those gene products that exhibit a strong (major) effect on the incompatibility of tissue between genetically non-identical individuals. In man—like mouse—some tissue antigens are very strong and others are very weak.

The strong transplantation antigens of man include carbohydrate antigens of red blood cells (ABH) blood group system and glycoprotein of the cell membrane (H LA system).

During experimental study of allograft rejection in mice, Groer (1930) observed two blood group antigen systems in mice —Antigen 1 common to all strains of mice; Antigen 2 present in certain strains of mice only which is related to allograft rejection and tumour immunity.

This was called H₂ antigen. In mice, H₂ antigen is major histocompatibility antigen coded for by a closely linked multi-allelic cluster of genes (H₂ complex) located in chromosome 17.

HLA Complex:

The human counterpart of H2 antigen system of mouse are alio antigens present on surface of leucocytes and are called human leucocyte antigens (HLA); and cluster of genes encoding for them is termed as HLA complex.

The term histocompatibility refers to the degree of compatibility or lack of it between the given tissues. By histocompatibility antigen is meant cell surface antigen that can evoke immune response to an incompatible host terminating in allograft rejection.

The HLA complex of genesis located in three regions on short arms of chromosome 6 and consists of eight genetic foci grouped in three classes. They are termed class I (HLA—A, B, C), class II (HLA—DP DO DR) and class III genes and their products provide a system for intercellular communication.

MHC antigens are essential for recognition of antigen by T-cells. They can only bind to antigen that are associated with these molecules. T helper cells with CD4 molecules on their surface recognise antigen in association with MHC class II molecules while T cells with CD8 molecules are restricted by MHC class I molecules. MHC class III products are important complement system and responsible for many cellular events.

Indications of HLA typing:

HLA antigens are widely distributed in tissues except red blood cells which contains only ABH antigens.

i. HLA system is very useful in tissue typing and matching prior to transplantation.

ii. It can be also used in paternity determination, anthropological survey and in establishing association between HLA and disease susceptibility.

iii. HLA-B27 is associated with ankylosing spondylitis, more frequently seen among the Caucasians (0.2%).

iv. Other diseases showing strong association with HLA include Reiter’s syndrome, juvenile rheumatoid arthritis, acute anterior uveitis, Coeliac disease and Grave’s disease.

HLA Typing:

i. Class I loci, HLA-A, B, C are detected by complement dependent cytotoxic reactions. Non-specific antiserum is collected from patients with whole blood transfusion and multiparous women who are often sensitized by foetal antigens. Monoclonal antibodies are now available. Lymphocytes of donor is typed against a panel of standard and recipient sera in presence of complement.

ii. Class II antigens are identified by mixed leucocyte reaction (M.L.R.). This is done by co-cultivation of donor and recipient lymphocyte to permit DNA synthesis. Enhanced DNA synthesis in the responder cells indicates more foreignness of class I MHC antigen.

iii. Preformed cytotoxic antibodies in patient’s sera is measured by mixing with donor’s lymphocytes and complement. Cytotoxic or cytolytic effect on donor’s lymphocytes indicates that recipient’s serum is reactive against the graft.