Locally produced cytokine and chemokines influence tissue-specific immune responses

Knowledge of the distinctive immunological characteristics of each tissue is only just emerging. Nevertheless a number of principles can be discerned:

Endothelium controls which leukocytes enter a tissue

Migration of leukocytes into different tissues of the body is dependent on the vascular endothelium in each tissue. For many years, it was thought that the endothelium in different tissues was essentially similar, with the possible exception of tissues such as the brain and retina, which have barrier properties (see below). Despite this common belief, it was well known that inflammation in different tissues had different characteristics, even when the inducing agents were similar.

It is now clear that a major element controlling inflammation and the immune response is the vascular endothelium in each tissue, which has its own characteristics; different endothelia produce distinctive blends of chemokines (Fig. 12.2). In addition the endothelium can transport chemokines produced by cells in the tissue from the basal to the lumenal surface by transcytosis, or by surface diffusion in tissues that lack barrier properties (Fig. 12.3).

Fig. 12.2 Production of chemokines by endothelium derived from different human tissues

Brain endothelium produces high levels of CXCL8 and CXCL10 associated with a TH1-type immune response. Dermal endothelium produces high levels of CCL5 associated with T cell migration, whereas lung endothelium produces high levels of CCL2, a chemokine that causes macrophage migration.

Fig. 12.3 Transport of chemokines

In most tissues, chemokines produced by cells in the tissue can move to the lumenal surface of the endothelium by transcytosis or by movement through the paracellular junctions, to be held on the endothelial glycocalyx. In tissues such as the brain, which have a barrier endothelium, there is limited transcytosis, and almost no paracellular movement of proteins such as chemokines. In these tissues, chemokine production by the endothelium is particularly important in controlling leukocyte migration.

The surface (glycocalyx) of vascular endothelium also varies considerably between tissues, and this affects which chemokines are retained on the lumenal surface, to signal to circulating leukocytes.

Hence the different sets of leukocytes present in each tissue can be partly related to the chemokines synthesized by the cells present, particularly the endothelium. For example, in normal lung there is a high level of macrophage migration, which relates to the high expression of CCL2 (macrophage chemotactic protein-1) by lung endothelium. In allergic asthma, the proportion of eosinophils increases, due to the production of IL-5 and CCL3 (eotaxin), which are characteristic of the TH2 response that tends to predominate in mucosal tissues (Fig. 12.4). By contrast, in the CNS lymphocytes and mononuclear phagocytes predominate in most immune reactions.

Fig. 12.4 Histological section of an airway from a case of fatal asthma

The lumen of an alveolus of the lung is heavily infiltrated with inflammatory exudates, fibrin, and cellular debris. Immunohistochemical staining with monoclonal antibody against EG2 indicates that the majority of cells are eosinophils.

(Courtesy of Arshad SH. Allergy: An Illustrated Colour Text. Philadelphia: Churchill Livingstone, 2002. With permission from Elsevier.)

Some tissues are immunologically privileged

There are certain sites in the body where fully allogeneic tissue can be transplanted without risk of rejection. These include the anterior chamber of the eye, the brain, and testes. Several factors may contribute to the immunological privilege of these sites, some of which affect the initiation of the immune response and some affect the effector phase.

The ‘privileged sites’ were long thought to be locations where adaptive immune responses are so dangerous that the immune system is either not allowed entry, is destroyed upon arrival, or is prevented from functioning. Recent evidence suggests that the concept of privileged sites may have been a misconception based on the limitations of the experimental assay systems. Once the experiments were expanded to include a wider variety of assays it became apparent that privileged sites are not immunologically impaired. They are simply sites that are able to promote certain kinds of beneficial classes of immune responses while suppressing classes that can do irreparable local damage. The mechanisms controlling immune reactions in the CNS are explored below.

The blood–brain barrier excludes most antibodies from the CNS

The blood–brain barrier is a composite structure formed by the specialized brain endothelium and the foot processes of astrocytes. Astrocytes are required to induce the special properties of brain endothelial cells, which have continuous belts of tight junctions connecting them to other endothelial cells (Fig. 12.5). An estimate of the tightness of the barrier is given by its trans-endothelial resistance, which is up to 2000 Ω/cm² in the CNS by comparison with values <10 Ω/cm² in most other tissues. In addition the brain endothelial cells have an array of transporters that allow nutrients into the CNS and a set of multi-drug resistance pumps that prevent many toxic molecules and therapeutic drugs from entering the brain. However, it is the very low permeability of the endothelium to serum proteins which is of particular interest for immunologists (see Fig. 12.5). For example, the level of IgG found in the CNS is normally approximately 0.2% of the level found in serum. The level may rise during an immune reaction as the endothelial barrier becomes more permeable in response to inflammatory cytokines. In some conditions, such as multiple sclerosis, there is often local synthesis of antibody within the CNS, which is reflected in abnormally high antibody levels in cerebrospinal fluid, even accounting for the increased leakage into the CNS. This finding demonstrates that some B cells have migrated into the CNS, and plasma cells have been identified in the spaces surrounding the larger blood vessels. Macrophages also contribute to immune reactions in CNS and they can synthesize some complement components locally (e.g. C3). However the overall level of serum proteins including antibodies and complement rarely exceeds 2% of the levels in serum even in the most severe inflammatory reactions.

Fig. 12.5 The blood–brain barrier

(1) Brain endothelial cells have a continuous ring of tight junctions, identified in this micrograph by the junctional marker ZO-1. The resulting permeability barrier excludes serum proteins from the brain tissue. (2) The graph shows the serum/cerebrospinal fluid (CSF) ratio for different proteins. There is an inverse relationship between molecular size and the level in CSF. Molecules such as transferrin, which are transported into the brain, are present at higher levels than would be expected from their size. Immunoglobulins are not transported.

Neurons suppress immune reactivity in neighboring glial cells

Researchers first examined single populations of cells from the CNS in vitro. For example, they found that astrocytes would respond to IFNγ by increasing their expression of MHC class I molecules and IFNγ would also induce the normally-absent MHC class II molecules. However, in vivo, astrocytes rarely respond in this way. It appeared that the local environment of the CNS could in some way suppress the ability of astrocytes to respond to IFNγ.

Subsequently it was found that when neurons are co-cultured in contact with astrocytes, then the ability to induce MHC molecules was suppressed, but if the cells were not in contact, they were not repressed (Fig. 12.6).

Fig. 12.6 Immunosuppression by neurons

The top graph is a FACS histogram which shows the expression of MHC class I molecules on astrocytes cultured in contact with (green) or separate from neurons (purple). Note that when neurons are in contact with astrocytes, the astrocytes have lower MHC class I expression. Staining of the negative control antibody is shown in blue. If IFNγ is added to the cultures (lower graph), astrocytes cultured alone (purple) increase their expression of MHC class I molecules, but those cocultured with neurons are still suppressed (green).

(Based on data from Tonsch U, Rott O. Immunology 1993;80:507.)

Subsequently it became clear that electrically active neurons are required. This means that neurons that are functioning normally can suppress their neighboring glial cells (and downregulate their own MHC molecules) whereas damaged neurons will lift the local immunosuppression to allow an immune response to develop.

Neurons can also act on microglia, which are resident mononuclear phagocytes of the CNS. The molecular mechanisms that underlie the suppressive activity of neurons include:

Immunosuppressive cytokines regulate immunity in the normal CNS

Observations of immune reactions in the CNS have led to the view that TH1-type immune responses with macrophage activation are generally most damaging, as are responses mediated by IL-17-secreting T cells (TH17 cells). By comparison TH2-type immune responses are generally less damaging, and strains of animals that make strong autoantibody responses against CNS antigens, are often less susceptible to CNS pathology than strains that make weaker antibody responses. Certainly, the production of IL-12, IL-23, and TNFα are associated with damaging responses in CNS. Interestingly IFNγ appears to have a dual role, involved in the acute-phase of CNS inflammation, but also necessary for the recovery phase (Fig. 12.7). Such observations have led to the view that immune deviation (the switching of an immune response from TH1 towards a TH2 type) can be protective in the CNS (see Fig. 11.2).

Fig. 12.7 Immune regulation in the CNS

In immune reactions in the CNS the myelin sheath formed by oligodendrocytes around nerve axons is particularly susceptible to damage by activated macrophages. Macrophages are activated by IFNγ produced by TH1 cells and memory T cells (Tm), and IL-12 is important in promoting this type of immune response. Alternatively TH17 cells induced by IL-23 and mononuclear phagocytes (macrophages and microglia) can also produce TNFα, which enhances macrophage activation and contributes to myelin damage. (Black arrows = production, green arrows = activation.)

This view is supported by findings that cells of the normal CNS can produce cytokines associated with TH2 responses. For example, astrocytes produce TGF-β, while microglia can produce IL-10 when cocultured with T cells, and both astrocytes and neurons secrete prostaglandins, which inhibit lymphocyte activation. Additionally, several neuropeptides and transmitters (e.g. vaso-active intestinal peptide, VIP) are suppressive. Acute immune responses can develop in the CNS, particularly in susceptible strains, but the normal immunosuppresive controls usually reassert themselves within 1–2 weeks, causing remission. Such a relapsing-remitting pattern of disease occurs in multiple sclerosis and the animal model of CNS inflammation, CREAE (chronic relapsing experimental allergic encephalomyelitis) (Fig. 12.8).

Fig 12.8 Chronic relapsing experimental allergic encephalomyelitis

The relapsing remitting disease course of CREAE in Biozzi Ab/H mice is shown in the days following immunization with myelin components on day 0 (black). Animals that were treated at day 7 with a cell line expressing a soluble TNF-receptor (to mop up TNFα in the brain) had a less serious acute disease and relapse.

(Based on data of Croxford et al. The Journal of Immunology 2000;164: 2776–2781.)

Immune reactions in CNS damage oligodendrocytes

Oligodendrocytes are glial cells that produce the myelin sheaths which act as electrical insulation around nerve axons. When immune reactions occur in the CNS these cells appear to be particularly vulnerable (Fig. 12.9). For example, multiple sclerosis is characterized by focal areas of myelin loss called plaques, typically a few millimeters in diameter. Nerve transmission through these demyelinated areas is seriously impaired, which may cause disease symptoms – weakness and loss of sensation. But it is only in the later stages of the disease that the neurons themselves are damaged.

Fig. 12.9 Type IV hypersensitivity in CNS

(1) The perivenous infiltrations of lymphocytes in parainfectious encephalomyelitis are confined to the area around the venule. There is little disruption of the tissue, but a stain for myelin shows that demyelination (2) extends out beyond the initial area of infiltration.

(Courtesy of Dr N Woodroofe and Dr H Okazaki.)

The reason that oligodendrocytes are vulnerable in multiple sclerosis is less clear. It is possible that they are most readily damaged, because they normally maintain very large amounts of plasma-membrane forming the myelin sheath. Another theory suggests that they are particularly susceptible to damage by serum molecules (e.g. complement), which leak into the CNS as a result of blood–brain barrier breakdown. Many researchers consider that they are targeted by autoantibodies which allow macrophages and activated microglia to recognize them, and that release of reactive oxygen and nitrogen intermediates from the phagocytes then damage the myelin.

The eye has powerful immunosuppressive mechanisms

Allogeneic corneal grafting is usually successful as a result of immune privelege, although rejection may occur – the eye has very limited self-regenerative capacity and can be completely destroyed by a cell-mediated immune response with the concomitant local production of TNFα and IFNγ (see Fig. 12.w1). The eye has therefore evolved several major mechanisms to actively suppress cell-mediated responses.

These cytokine and molecular controls on immunity are underpinned by the cellular organization of the eye, which has barriers that limit the movement of molecules into the retina, anterior chamber, and vitreous (Fig. 12.w2). As with the CNS, it is the combination of immunosuppressive and tolerogenic mechanisms which normally maintains immune privelege in the eye.

Fig. 12.w2 Anatomic and immunological basis of immune privilege in the eye

The uvea, consisting of the iris, ciliary body, and chorea, is highly vascularized, but lacks draining lymphatic vessels. Tight junctions between the vessels in the iris and between non-pigmented ciliary epithelial cells maintain a blood–aqueous barrier. Similar junctions on the retinal pigment epithelium and the retinal endothelium maintain a blood–retinal barrier. The iris and ciliary body secrete immunomodulatory cytokines.

Local immune privilege may extend systemically

In experiments in mice, where allogeneic tumors were transplanted into the eye, it was found that the recipients became more generally tolerant of grafts which shared the tumor antigens, regardless of the site of transplantation (Fig. 12.w3).

Fig. 12.w3 Tolerance to allografts transplanted into the eye

Tumor cells of tissue type A transplanted into the eye are tolerated. Subsequently skin grafts of tissue type A × B are also tolerated in these animals. Tolerance to A, induced within the eye, extends to other tissues, and can turn off the normal immune response that would be expected against a type B skin allograft.

Following up on this finding, it was discovered that spleen APCs that had been incubated with fluid from the anterior chamber of the eye were no longer able to induce T cell proliferative responses, but instead promoted production of IL-4, IL-10, and TGFβ. This switch in immune response was termed anterior chamber associated immune deviation (ACAID) and it illustrates that the way an immune response that develops in one tissue can have more generalized effects.

Altogether, the picture that emerged is of an organ that actively promotes a type of immunity that is protective and beneficial (the IgA response) while suppressing a type

The gut immune system tolerates many antigens but reacts to pathogens

There are many examples, where an individual encounters an antigen in food, and subsequently becomes tolerant to it – they do not make an immune response when the antigen is subsequently given in an immunogenic form. This phenomenon is called oral tolerance and it is related to nasal tolerance where antigen delivered to nasal mucosa as an aerosol inhibits subsequent immunization.

Oral tolerance, and the related phenomenon of nasal tolerance, illustrate two points:

Tolerance is not the only form of immunity that arises from ingestion of antigens. Oral vaccination has been recognized since 1919 when Besredka noticed that rabbits were protected from fatal dysentery by oral immunization with killed shigellae. The attenuated polio vaccine that was developed in the 1950s was also given as an oral vaccination. In both cases these vaccines are not viewed as harmless antigens by the immune system:

Gut enterocytes influence the local immune response

Intestinal epithelial cells (enterocytes) are the major cell-type forming the epithelium of the gut. These cells have continuous tight junctions and thus form a barrier to prevent antigens from entering the body. The enterocytes of the gut considerably influence the local immune response by secreting a variety of immunomodulatory factors such as TGFβ, VIP, IL-1, IL-6, IL-7, CXCL8, and CCL3. The Paneth cells at the bottoms of the crypts meanwhile produce several natural antibiotic peptides (Fig. 12.11), which help prevent bacterial overgrowth in these sensitive sites of cellular differentiation.

Fig. 12.11 Structure of the villi and crypts of the small intestine

Stem cells give rise to Paneth cells at the base of the crypt, which secrete antimicrobial peptides. The enterocytes are derived from the rapidly dividing transit-amplifying population and will eventually be shed from the tips of the villi. These cells secrete immunomodulatory cytokines.

Intra-epithelial cells (IEL) and some dendritic cells are located in the epithelial layer and enterocytes may promote their migration. More lymphocytes and phagocytes are found in the lamina propria, which lies beneath the epithelium.

By expressing E–cadherin, a ligand for αEβ7-integrin, and by secreting TGFβ, which upregulates the expression of αEβ7, enterocytes may provide a signal for the selective accumulation of IELs that express this integrin. In this way, the tissue cells invite the residency of cells that promote certain types of immunity and aid in repair.

The major areas of gut-associated lymphoid tissue (GALT) include the Peyer’s patches in the small intestine, the lymphoid follicles throughout the gut, and the mesenteric lymph nodes which receive lymph draining from the intestinal villi and the other lymphoid tissues (see Fig. 2.53). It is worth noting that food antigens are primarily present in the small intestine, whereas bacterial antigens are more prevalent in the large intestine.

IELs produce many immunomodulatory cytokines

Tolerance to food antigens depends on a variety of overlapping mechanisms, but the production of TGFβ and IL-10 are particularly important. For example, IL-10 knockout mice develop colitis, if they contact appropriate triggering micro-organisms and in humans, IL-10 is a susceptibility locus for ulcerative colitis. TGFβ is an important element in the TH2 spectrum of cytokines and also induces FoxP3 in naive T cells, thus promoting development of regulatory T cells. Tregs, which are abundant in the lamina propria also produce IL-10 and TGFβ, which further limit inflammatory reactions in the gut (Fig. 12.12). It is not clear exactly how Tregs are stimulated within the gut; they express high levels of TLRs, particularly TLR2, 4, and 8, so they can respond to the high potential load of MAMPs, but they clearly respond differently to APCs and effector CD4⁺ T cells.

Fig 12.12 Immune cells in the intestine

Immune cells in the intestine include the intra-epithelial γδ T cells, effector αβ T cells and dendritic cells found in the epithelial layer. The lamina propria includes populations of macrophages, effector T cells and regulatory T cells (Tregs). IgA-producing B cells and associated T cells are found in the encapsulated lymphoid organs, including the Peyer’s patches and the lymphoid follicles. Tregs are also found in T cell areas of the lymphoid tissues.

When immune responses develop in the gut, IELs can produce IL-1, lymphotoxin (LT), IFNγ, and TNFα, so a switch in the balance of pro-inflammatory cytokines and regulatory cytokines in the epithelium and lamina propria occurs when damaging immune reactions develop in the gut.

Many IELs have an activated or memory phenotype and recognize the ancient conserved MHC class I-like molecules, MICA and MICB, which are upregulated by cellular stress. When activated by MICA and MICB some of the mucosa-associated lymphocytes secrete epidermal cell growth factor (ECGF) and may therefore function to repair and renew damaged intestinal epithelial cells.

Hence, by expressing molecules, the tissue cells can activate their local resident lymphocytes regardless of the specific antigen that is the target of the immune response. In this way the tissue can influence local immunity against many different antigens.

Chronic inflammation in the gut

The most common types of chronic inflammatory disease of the gut are inflammatory bowel disease (IBD) including Crohn’s disease, which may affect the entire intestinal tract, and ulcerative colitis affecting the large intestine. Both of these conditions are thought to occur because of an over-reaction to intestinal bacteria; both have a strong genetic component and pattern recognition receptors (e.g. NOD2) are disease susceptibility loci.

In celiac disease, the subject is sensitive to gluten in the food, and susceptibility is strongly related to HLA haplotype and to loci for inflammatory cytokines and chemokines. In all three conditions, ulcerative colitis, celiac disease and Crohn’s disease, there is production of TNFα and IFNγ, dysregulation of the normal mucosal immune response and damage to tissue, including the epithelium responsible for absorbing nutrients.

The chronic inflammatory diseases all appear to be due to a reduction of the normal tolerogenic mechanisms in the gut, so that the immune response is tipped towards inflammation.

Immune responses in the lung

The lung is another example of a tissue that is in normal contact with external antigens, and pathogens, including bacteria from the upper airways. Particles trapped in the trachea and on the bronchial mucosa are propelled upwards on the ciliary escalator, and this considerably reduces the antigen load reaching the lung. Bronchioles and alveoli contain large numbers of pulmonary macrophages, while the lung tissue also contains lymphocytes and respiratory (plasmacytoid) dendritic cells (pDCs) (Fig. 12.w4). The macrophages and pDCs are major sources of IFNα, and IFNβ, which are particularly important in controlling the initial spread of viral infections.

Fig. 12.w4 Immune responses in the lung

In the resting lung memory T cells and macrophages are present in the alveolar air spaces and the lung tissue. Respiratory dendritic cells (DCs) are located below the respiratory epithelium which consists of type-I pneumocytes with a large surface area for gas exchange, and type-II pneumocytes which secrete surfactants. In acute inflammation NK cells and neutrophils enter the air spaces. How the inflammatory response develops depends on the initiating pathogen. Viral infections result in the accumulation of effector T cells and macrophages, whereas neutrophils persist in many bacterial infections.

Early recruitment of leukocytes to the lung is mediated by chemokines secreted by the macrophages, pDCs and the epithelial cells of the lung itself (pneumocytes). Neutrophils and NK cells are the first cells to appear after infection, while dendritic cells traffic to the local bronchus associated lymphoid tissue (BALT). Migration of the dendritic cells normally occurs at a steady rate, but following infection there is an increase in movement of pDCs to the lymphoid tissues and they increase their expression of MHC class-II and costimulatory molecules B7 and CD40.

The stucture of BALT resembles that of other encapsulated lymphoid tissues, with distinct T and B cell areas, and high endothelial venules and the migration of the pDCs to the local lymph nodes depends on signaling from CCL21 acting on CCR7, as in other tissues.

In viral infections of the lung, the early recruitment of NK cells, which recognize virus-infected cells via NCR1, helps control the spread of the infection, until the later arrival of CD4⁺ and CD8⁺ effector T-cells; CD8⁺ CTLs recognize and destroy virally-infected pneumocytes using Fas/FasL, and by releasing granzymes and perforin.

In bacterial infections, neutrophils and macrophages are prevalent in the bronchi (bronchitis) or the alveoli (pneumonia), and the smaller air-spaces fill with a fluid exudate that leaks through the damaged pulmonary epithelium.