Innate immunity

There are two systems of immunity, innate and adaptive, coexisting to keep pathogens out. Innate immunity, phlyogenetically earlier, is nonspecific and immediate in response. It is the first immune response mounted against invading pathogens. The key components of innate immunity are a cadre of leukocytes and plasma proteins that are capable of detecting and destroying pathogens. These leukocytes include polymorphonuclear cells, monocytes, macrophages, natural killer (NK) cells, eosinophils, and basophils and the plasma proteins belong to families of the complement cascade, clotting cascade, and acute-phase reactants.

Adaptive immunity

In contrast, adaptive immunity, as the name implies, is antigen-specific, adaptive, transferable, and has other cardinal features like immunologic memory and tolerance. Adaptive immunity has evolved and developed novel molecules like the antibody and the T-cell receptor (TCR), which come in almost limitless varieties to recognize most molecules of biological interest, including both self and nonself. These molecules, developed in the absence of exogenous stimulation, have unique configurations that confer specificity in antigen recognition. The key cellular components of adaptive immunity are the T and B lymphocytes which possess unique receptors for recognizing billions of different antigenic epitopes.⁸ Each clone of lymphocyte expresses molecularly identical receptors on the surface; hence, in order to recognize billions of different epitopes, there are billions of unique lymphocytes. Upon maturation in the thymus (T cells) and the bone marrow (B cells), cells remain quiescent in G0 of the cell cycle until they encounter the complementary antigen of sufficient affinity to their receptors. Binding with the receptors in the presence of appropriate costimulation is the initiating event to trigger an immune response targeting elimination of the complementary molecule. If this molecule is pathogen-derived, activation will lead to the elimination of the pathogen, but if the molecule is self-derived then activation will potentially lead to autoimmunity, tissue injury, disease, and the destruction of host tissue. Activation of naive lymphocytes results in the emergence of clones of lymphocytes through proliferation, each with a unique receptor to recognize one epitope of the inciting antigen. The cytokine environment during activation of naive T cells is responsible for differentiation into three major types of effector T cells. Interferon (IFN)-γ leads to Th1 cell production, while interleukin (IL)-4 is responsible for Th2 production. Some Th1 and Th2 cell clones will become memory cells which are responsible for the enhanced immune response if the antigen is re-encountered. The rest of Th1 will secrete cytokines targeted at macrophages and other cells mediating cellular immunity. By contrast, Th2 will stimulate B cells into proliferation and antibody production, mediating humoral immunity. Th17 are a recently discovered family of T helper cells that are capable of producing IL-17. IL-1 and IL-23 seem to be needed for human Th17 differentiation.⁹ Th17 play an important role in combatting various bacterial and fungal species by producing IL-23. In contrast, large amounts of Th17 can be found in autoimmune diseases like uveitis, multiple sclerosis, and rheumatoid arthritis.¹⁰

Both immune and accessory cells, including those of the innate immunity system, are activated to eliminate pathogens. In this way, adaptive immunity serves to enhance immune protection through better-coordinated and more specific attacks using innate immunity mechanisms. Within the genome lies the ability to create antibodies and TCR for antigens that bind to self antigens with high affinity.¹¹ Hence, the threat of autoimmunity is inborn and regulation is crucial. The ability to distinguish between foreign and self, and the ability to regulate autoimmunity, are critical for survival.

The immune response can be seen as analogous to the neural reflex arc, which has an afferent limb, a central process, and an efferent limb. In the immune afferent limb, antigens are captured, processed, transported, and finally presented to the lymphocytes. As the naive lymphocytes can only be activated in the organized lymph tissues in secondary lymphoid organs such as lymph nodes, spleen, tonsils, and Peyer’s patches, it needs specialized cells to execute the immune afferent limb. These cells are professional antigen-presenting cells (APCs). Dendritic cells and macrophages, both bone marrow-derived, have APC functions serving to capture antigens through phagocytosis and endocytosis, process the antigens, and present the processed antigen in conjunction with special major histocompatibility complex (MHC) molecules on their cell surface. In addition, the APCs provide the necessary costimulation needed for lymphocyte activation by upregulating an array of surface molecules (CD80, CD86, intercellular adhesion molecule-1, lymphocyte function-associated molecule-3, and CD40) that function as ligands for receptors expressed by the lymphocytes. They also secrete cytokines such as IL-12, IL-6, IL-10, and IL-1β serving similar costimulation functions. Costimulation is required as the second signal for full activation of the lymphocytes, independent of the first signal, which is the antigen and the lymphocyte receptor engagement.¹² There are differences in antigen presentation to B and T lymphocytes. For B lymphocytes, the receptor, which is a surface-bound antibody, can engage a naive antigen directly while TCRs can only recognize peptide fragments presented on special surface molecules (MHC classes I and II). MHC class I, which is present in most cells, presents peptides derived from protein degradation in the cytoplasm (intracellular antigens such as viral and intracellular microbe products), while MHC class II molecules, which are present in APCs and lymphocytes, present peptides from phagocytic vesicles (extracellular antigens from the microenvironment).

Upon activation, the first cells to respond are the CD4⁺ T cells specific for the presented peptide on the MHC class II molecules of the APCs. The CD4⁺ lymphocytes will proliferate and secrete an array of cytokines, including IL-2, IL-3, granulocyte–macrophage colony-stimulating factor, IFN-γ, and IL-4. These cytokines serve as growth and stimulation factors for the lymphocytes and APCs, hence amplifying the proliferation process. In addition, IL-2, IFN-γ, and IL-4 also promote the differentiation of CD8+ T lymphocytes to mature cytotoxic T lymphoctes (CTLs) which recognize antigen-derived peptides presented on class I MHC molecules. Upon activation, these CD8+ CTLs function as effectors to lyse the targeted cells and also produce proinflammatory cytokines, especially IFN-γ. The cytokine production by CD4⁺ T cells also promotes activation and differentiation of B lymphocytes. IFN-γ and IL-2 stimulate B cells to produce complement-fixing immunoglobulin G (IgG) antibodies, while IL-4, IL-5, IL-6, and IL-10 result in the production of noncomplement-fixing IgG, IgE, or IgA antibodies. The final products after the central processing in the lymphoid tissues are immune effectors. These are the CD4⁺, CD8⁺ T cells and B cells that have receptors specific for the inciting antigen. They are transported, predominantly via a hematogenous route, to the site of the inciting antigens to execute their effector function, thereby completing the efferent loop of the immune response. At the target sites, sites of infection and inflammation, vessels are leaky, the vascular endothelial cells display ligands that bind to receptors on the immune cells, and chemokines secreted by the local inflammatory cells serve to attract more immune effector cells to the site. While the engagement and activation of B cells can be direct, activation of the T cells still needs the APCs for antigen recognition. The activated CD4⁺ T cells secrete cytokines such as IFN-γ and tumor necrosis factor (TNF)-α which in turn attract and recruit the cells of the innate immune system, such as monocytes, macrophages, and NK cells, to the site to effect the actual destruction of the antigen or pathogen through generation of cytotoxic products and phagocytosis of the pathogens. B cells secrete antibodies specific for an antigen which will result in direct killing/lysis of the target and also recruit polymorphonuclear cells via complement activation and the complement activation products respectively. In this way, the adaptive immune system serves to direct the innate immune system components to target the inciting antigen or pathogen.

Immune regulation

The immune system can be regulated at any part of the immune response loop, i.e., afferent limb, central process, and efferent limb. Regulation of the autoimmune response can be via any of the following mechanisms: central tolerance – clonal deletion and peripheral tolerance – clonal anergy, T-cell suppression, immune deviation, immunologic ignorance, and antigen sequestration. Since the genome has the capability to generate both self- and nonself-recognizing antibodies and TCRs, mechanisms must be in place to contain and prevent the activation of self-reactive T cells. During lymphocyte development, both T and B cells with receptors that recognize self molecules with high affinity are clonally deleted via apoptosis (central tolerance).¹³ However the process is not foolproof, as autoreactive T and B cells in the periphery exist even in normal individuals. Many tissue-specific antigens, like the eye-restricted molecules, may not be expressed in the thymus or cells may have escaped the selection process by the central tolerance mechanism in the thymus without encountering the specific self antigen. Hence, the potential for induction and expression of autoimmunity still exists, and mechanisms to contain these autoreactive immune cells are crucial to prevent autoimmunity. Evidence for peripheral tolerance can be found in all phases of the immune response, i.e., afferent limb, central process, and efferent limb.^14,15 The afferent limb depends heavily on the functional properties of the APCs which offer many opportunities for modulation. First, the antigen can be sequestrated and prevented from contact with the APCs by physical barriers or by rendering APCs incapable of antigen capture in the microenvironment (sequestration). Second, the ability of the APC to degrade, process, and express the antigens can be inhibited. Third, the antigen-bearing APCs may be prevented from migrating to the lymphoid tissues. Lastly, the ability of the APCs to generate the costimulatory signal can be inhibited or different costimulatory signals may be produced (immune deviation).

Regulation of APC function can be seen as a form of immunologic ignorance where reactive autoimmune cells are present but never have the opportunity to encounter its antigen in a proper immunogenic form due to a deficiency in antigen presentation. In the central processing phase of the immune response, two signals are required for full activation of the lymphocyte. The absence of costimulation in the presence of antigen-specific stimulation renders the T cells unresponsive to further antigen stimulation, even in the presence of costimulatory signals (anergy).¹⁶ On the other hand, too much costimulation in the presence of high antigen dose may lead to profound cell activation and apoptosis. Both mechanisms will result in tolerance.

It is also known that APCs from certain immune-privileged sites such as the eye, and APCs in the gut processing orally ingested antigens, may cause some immune cells to develop into immunoregulatory cells like IL-10-secreting Tr1 cells or transforming growth factor-β (TGF-β) secreting Th3 cells. These cells have in common that they are adaptively regulatory. They receive a suppressive potential following specific antigen stimulation in a particular cytokine milieu.¹⁷

In the efferent limb, immune regulation can occur via targeting at the level of antigen-specific immune effector cells. One such mechanism would be through Fas ligand – Fas-induced apoptosis of lymphocytes.^18,19 The immune-reactive lymphocytes express the CD95 receptor (Fas receptor). On encountering the Fas ligand, which is expressed in immuno-privileged eye tissues such as the cornea and retina, these immune cells undergo apoptosis. Other molecular mechanisms, such as TNF-α production by immune and accessory cells, such as Müller cells and retinal pigment epithelial (RPE) cells during antigen encounter, can also result in apoptosis, thereby deleting these immune-reactive cells. Similar to the central process, effector cells need costimulatory signals to be activated and the expression of inappropriate costimulatory signals such as IL-10 or TGF-β by APCs can lead to anergy or reprogramming of lymphocytes into immunoregulatory cells secreting immunosuppressive cytokines, thereby further suppressing autoimmunity.^20,21

An important regulator of immune responses is the CD4⁺ CD25high forkhead box protein 3 (Foxp3)+ regulatory T cell (Treg). There are two groups of Treg in humans: the naturally occuring Treg and the induced Treg. The natural Treg is developmentally determined in the thymus as a distinct T-cell subpopulation that is specialized for suppressive function.¹⁷ It is distinguished from the induced Treg by a demethylated promoter region for Foxp3.²² Foxp3 seems to act as a repressor of transcription that regulates T-cell activation. A dysfunction of Foxp3 results in hyperactivation of T cells and severe autoimmune disease.²³ Natural and induced Tregs maintain peripheral tolerance by modifying the functions of other T cells, both CD4⁺ and CD8+ populations, either directly through T-cell to T-cell interactions, or indirectly through APCs.²⁴ Hence they are crucial for maintaining immune homeostasis and controlling autoimmunity in order to keep the immune system healthy.

Blood–retinal barrier in diabetic retinopathy

The initial disorder causing both nonproliferative and proliferative diabetic retinopathy is chronic hyperglycemia leading to retinal hypoxia. Compensatory mechanisms like vasodilatation are a regulatory attempt to increase retinal blood flow. After exhaustion of autoregulation, a disruption of endothelial tight junctions in the retinal vasculature can occur.⁴ Furthermore, chronic hyperglycemia seems to be able to induce a hypoxia-mediated expression of growth factors and cytokines in the retinal endothelium, such as insulin-like growth factor-1, vascular endothelial growth factor-A, angiopoietin-2 and TNF-α, leading to leukocyte adhesion, endothelial cell injury, increased permeability of endothelial cells, and finally angiogenesis.^31–33 The resulting plasma leakage from the inner blood–retinal barrier may cause diabetic macular edema and consecutive vision loss.

Transportation of antigens

The blood–retinal barrier serves to keep tissue-specific antigens sequestrated. There is an absence of lymphatics in the retina, although there is evidence that antigen may still be able to be transported to the lymph nodes.⁴¹ Tissue drainage via the hematogenous route may alter the APC function.⁴² There is also a reduced expression of MHC class I and II molecules and an absence of bone marrow-derived cells that function within tissues as APCs in the normal retina.⁴³

Downregulatory immune environment

The DIE is an evolutionary adaptation of the eye to protect itself against excessive inflammation. A small focus of inflammation in the eye has a far greater impact than a similar one in the kidney. The posterior segment seems to have several active factors that help to keep the local immunoenvironmental balance in a downregulatory state. Active factors include the downregulatory effect of Müller cells in cell-to-cell contact with lymphocytes.⁴⁴ Furthermore, there are many other ocular cells that affect DIE (NK T cells, RPE, microglia, F4/80⁺