Michael J. Day, BSc, BVMS (Hons), PhD, DSc, DECVP, FASM, FRCPath, FRCVS
School of Clinical Veterinary Science, University of Bristol, Langford, United Kingdom
The Mucosal Immune System
The mucosal surfaces of the body include those of the respiratory, gastrointestinal and urogenital tracts, the conjunctivae and mammary glands. These surfaces and the skin are the most common routes of entry for potential pathogens into the body. Consequently, the mucosae are richly endowed with immune defenses such that, for example, the intestinal tract has the highest concentration of lymphoid tissue of any anatomical location. The unique nature of mucosal barriers means that they have evolved specific immunological defense mechanisms - many of There are elements of both innate and adaptive immunity at each mucosal barrier. Innate immunity is a simpler, evolutionarily older, non-specific form of protection that is constantly present to provide immediate 'first line' defense from pathogens. Innate immune defenses include various physical properties of the mucosal barriers, for example the respiratory tract 'mucociliary escalator' or the peristaltic movement of the intestinal tract. Although some mucosal surfaces are relatively sterile, others are endowed with a rich microflora that provides competition for space and nutrients to any potential pathogen. At most mucosal surfaces, external antigens and potential pathogens are prevented from accessing the body by a single-layered epithelial barrier. This is generally bathed in glandular secretion containing an array of antimicrobial substances such as polyreactive immunoglobulin, complement molecules, enzymes such as lysozyme, and antimicrobial peptides such as defensins. Polyreactive immunoglobulins are generally of the IgA and IgM classes and have low affinity antigen receptors capable of binding to an array of microbial epitopes. The innate immune system also includes a range of leucocytes resident within the epithelial barrier or underlying lamina propria that function non-specifically to provide rapid immune protection. Two populations may be found within the epithelial lining - dendritic antigen presenting cells that form the bridge between the innate and adaptive immune system, and in some locations (particularly the intestine) a population of T lymphocytes that express the γδ form of the T-cell receptor. Other innate immune cells include tissue macrophages and mast cells and natural killer (NK cells). Although not normally resident, granulocytes (neutrophils and eosinophils) can be rapidly recruited to mucosal sites when required. The adaptive immune system of mucosal surfaces is relatively specialized and includes the range of T and B lymphocytes, plasma cells, cytokines and specific immunoglobulins. Normal mucosal surfaces are continually accessed by recirculating pools of lymphoid cells that undertake the process of 'immune surveillance'. These cells may be diffusely scattered throughout the lamina propria, may form isolated lymphoid follicular aggregates or more organized lymphoid areas such as the intestinal Peyer's patch or regions of bronchial associated lymphoid tissue (BALT). The best described of these structures is the Peyer's patch which is a non-encapsulated focus of B-cell follicles surrounded by an intervening T-cell zone. The epithelium overlying the Peyer's patch is modified (dome shaped) and includes the antigen-sampling microfold cells (M cells) that capture luminal antigen and transfer it to the underlying lymphoid tissue. Dendritic cells within the villous lamina propria can also sample antigen by extending their dendritic processes between enterocytes. Lymphatic drainage connects the mucosal lymphoid tissue to the regional (e.g. bronchial or mesenteric) lymph nodes that are the likely site of generating the adaptive immune response. The effector populations of the mucosal adaptive immune system include both Th1, Th17 and Th2 CD4+ lymphocytes, CD8+ cytotoxic T cells, plasma cells (primarily committed to the production of IgA and IgG) and importantly, populations of regulatory T cell (e.g. Th3 cells, Treg or Tr1 cells) that are responsible for preventing abnormal immune responses to harmless dietary antigens or antigens derived from the resident microflora. Failure of the action of these regulatory populations underlies many of the inflammatory enteropathies of man and animals. Many of these T cells perform their role by secreting soluble messengers (cytokines and chemokines) that regulate the function of the leucocyte populations of the mucosal surface.
A unique feature of the adaptive immune response of mucosal surfaces is the 'common mucosal system' that enables recirculating lymphocytes activated in the context of one mucosal surface to access other mucosae by virtue of shared adhesion molecule expression. Some adhesion molecules however are responsible for selective 'homing' of recirculating cells to the tissue of origin - for example combination of endothelial MAdCAM and lymphocyte αβ allows selective egress of cells into the intestinal lamina propria. One further significant part of the mucosal adaptive immune response is antigen-specific IgA. Local tissue plasma cells secrete the dimeric form of this molecule which is captured by the polymeric immunoglobulin receptor (pIgR) expressed on the basolateral surface of enterocytes. The IgA-pIgR complex is internalized by the enterocyte and re-expressed on the luminal surface, from which the IgA is released, carrying with it a portion of the pIgR that forms the protective secretory component. IgA can capture luminal antigen and recycle back through the enterocyte to the lamina propria side of the barrier.
Measurement of Mucosal Immunity
Measurement of mucosal immune function is technically challenging and not often performed diagnostically in veterinary medicine. However, at the research level, a number of tools have been developed to allow us to assess aspects of mucosal immunity in normal and diseased animals. Assessment of whether mucosal surfaces are able to produce adequate concentrations of IgA has always been a fundamental measure of their immune function. It was previously thought that as most circulating (blood) IgA is derived from mucosal surfaces, measurement of serum IgA concentration would provide an indirect measure of mucosal IgA secretion. Serum IgA is readily detected in the dog by the application of commercially available single radial immunodiffusion test kits, although these differ in quality between manufacturers. However, studies in which serum and mucosal IgA concentrations have been compared suggest that there is relatively poor correlation between these two parameters, and moreover, at least in the dog, there is both day-to-day and diurnal variation in serum IgA concentration. Therefore, the most precise means of determining mucosal production of IgA (and also IgG and IgM) is to measure concentration in a specific mucosal secretion. This provides two challenges - firstly in obtaining such secretions, freezing them rapidly to avoid proteolytic degradation (or adding anti-proteolytic substances) and accounting for the dilution effect determined by the volume of secretion, and secondly in devising a sufficiently sensitive assay for detection of the relatively low levels of immunoglobulin found within secretions. Despite these challenges, it has been possible to harvest a wide range of secretions from dogs and cats for study (e.g. tears, saliva, bile, colostrum, milk, duodenal juice, faecal extracts) and to normalize immunoglobulin content relative to other proteins (e.g. albumin). Detection of these immunoglobulins requires the development of capture ELISAs and these have been produced and validated for both the dog and cat. In the dog, it has also been possible to detect the secretory component and develop assays specific for secretory IgA. In studies related to the efficacy of intranasal vaccines it has proven possible to measure antigen-specific antibody production locally and systemically following such challenge. Allergen-specific IgE has also been measured in, for example, faecal extracts in cases of dietary hypersensitivity or in broncho-alveolar lavage fluid in cases of respiratory allergic disease. An alternative to measuring immunoglobulin in secretions has been the establishment of explant cultures of mucosal tissue and assessment of the in vitro synthesis and secretion of immunoglobulin into the tissue culture medium. It has proven possible to establish such explant cultures of canine intestinal mucosa and to keep these alive for up to 72 hours whilst monitoring the progressive increase in IgA secretion following de novo synthesis.
A greater challenge is to assess cellular (as opposed to humoral) aspects of mucosal immunity. One means by which this is achieved is to collect biopsies of the tissue of interest and undertake immunohistochemistry to identify and enumerate subpopulations of lymphoid cells. In dogs and cats this has been done using a range of markers able to detect CD4+ and CD8+ T cells, B cells, plasma cells and antigen presenting cells. It is also possible to take fresh biopsy samples and to tease them apart by enzymatic degradation to release leucocytes into cell suspension. These cells can be similarly immunolabelled and enumerated by the use of a flow cytometer. This technique has been applied to both canine and feline intestinal tissue. Theoretically such cell suspensions could also be used for determining the function of the extracted cells in various types of proliferative assay but this has not yet been described for companion animals. In addition to enumerating leucocyte populations an indirect measure of their function is by the detection of cytokine and chemokine proteins produced by these cells within the mucosal microenvironment. Reagents designed to do this have only just become available and thus far been applied only to serum samples or cultures of blood lymphocytes. The more widely applied methodology for determination of cytokine production within tissue has been the use of real-time reverse transcriptase polymerase chain reaction (RT-PCR) to quantify cytokine and chemokine gene expression (transcription) within the tissue of interest. Gene transcription is generally presumed to correlate with eventual protein synthesis.
Diseases with Possible Defects in Mucosal Immunity
A number of the canine primary immunodeficiency disorders are believed to have an underlying defect in mucosal immunity that predisposes affected animals to chronic recurrent mucosal infections. None of these disorders have yet been associated with a well defined genetic mutation and so remain putative immunodeficiencies at this time. As our ability to measure mucosal immune function is limited, most of these diseases are presumptively associated with inadequate mucosal production of either (or both) IgA and IgG.
A list of further reading can be supplied on request.