Click here for the PDF version

Contents

The interaction with the human body and its environment is very elaborate. The whole body is covered with epithelium that mainly functions as a barrier. On mucosal surfaces this barrier is very delicate. Epithelial cells are the first cells that encounter infectious bacteria and as such, they have developed several mechanisms for microbial protection. Its function is not only protection against the environment but also interaction with the environment.

At sites where a big load of incoming antigen and pathogens have their first contact with the body, mucosal tissues show important lymphoid differentiation. It is well known that palatine and nasopharyngeal tonsils, because of their unique structure, have an important role in the immunologic surveillance and maturation of both humoral and cell-mediated immune response. This nasal associated lymphoid tissue (NALT) has characteristics that can also be found in other areas of the body (BronchialALT, GutALT). These organized mucosal lymphoid tissues have a role in transport, processing and presentation of antigens to the immune system. The subepithelial connective tissue contains elaborate mucosal cells with direct or indirect effector functions: terminally differentiated B cells, cytotoxic T cells, NK cells, and antigen presenting cells such as macrophages and dendritic cells. But how do these cells get their information? When we presume that important antigen passage through the epithelium is possible without endangerment of its barrier function, we can conclude an important role for mucosal epithelium in antigen processing.

The mucosal associated lymphoid tissue, also called MALT, contains specific epithelial cells with an important antigen presenting function, the so-called M cells. These cells, with microfolds instead of microvilli and a marked undulating cell surface with large basolateral pockets containing lymphoid cells, have an important capacity for transepithelial transport and delivery of foreign antigens and microorganisms to the underlying lymphoid tissue. The expression of distinct glycoconjugates by M cells in various tissues and species suggests an important role for carbohydrate epitopes in the function of this unique cell type. The structural modifications of the M cell apical surface and the display of oligosaccharides together allow M cells to facilitate adherence, uptake, and immunological sampling of m.o. But their immunological features do not argue a selective binding of antigens or pathogens. Therefore, M cell transport is a double-edged sword: certain pathogens exploit the features of M cells that are intended to promote uptake for purpose of immunological sampling.

We can conclude that M cells are, in the important tissues of the MALT, essential for unbiased presentation of non-self-matter to the adaptive immunity, which is crucial for the integration with the environment. These M cells might have a role in enhanced delivery of mucosal vaccines in human but further research is needed to establish this. But M cells represent only a small percentage (<5%) of follicles associated epithelial cells. The mucosal surfaces of the respiratory and digestive systems are mainly covered by delicate undifferentiated epithelial barriers were only a single layer of epithelial cells separates the outside world from interstitial tissues. Are these epithelial cells capable of protecting themselves and do they have a role in antigen presentation?

Millions of years of co-evolution and co-existence of both host and pathogens created a balance between them so they can both survive without too much energy spent on their defense. Knowing that bacteria grow fast (a bacterial population doubles in 20 min) and that small species do not have an adaptive immunity to protect them, these species need a fast way to eliminate microorganisms before they are overgrown by them. Also humans, with a slow adaptive immune system that needs time to expand clonally, could have use of a fast defense mechanism. Specific antigen recognition by lymphocytes is limited during the initial encounter with microorganisms (m.o.) and is mostly effective against persistent microbes or against microbes previously encountered by the host. The host resistance during the early phase of interaction with microbes does not depend on this specific antigen recognition.

In humans, different mechanisms developed to give an early protection against invading m.o. In the upper airways, under normal conditions the contribution of mechanical factors such as mucociliary clearance, phagocytic clearance, or sneezing may shorten the time available to microbes to adapt to the deleterious effect of nasal secretions. In 1922, Alexander Fleming discovered antimicrobial properties of nasal fluid that he attributed to lysozyme. Since then, there have been few studies characterizing the innate immune function of nasal fluid. Several groups have attributed the microbicidal activity of nasal fluid mostly to two major components, lysozyme and lactoferrin. Before Fleming, Metchnikoff surmised that microbicidal substances must be present in phagocytes, because immediately after breaching epithelial surfaces mobile phagocytes are able to ingest and kill the invaders.

Due to the discovery and development of penicillin and other antibiotics and their important therapeutic impact in the first part of last century, the interest for natural antimicrobial components was very meager. The resistance problem and the discomfort for using these chemotherapeuticals, especially in children, renewed the interest for innate, natural defense mechanisms.

Probiotics that maintain a protective microbial flora and soluble oligosaccharide anti-adhesive therapeutic agents that prevent binding of microorganism's carbohydrate-binding proteins with membrane-bound oligosaccharides on human cells, are some of the possible antimicrobial therapy strategies with no risk of resistance. But do we need to use additional strategies to protect our surfaces against m.o.? Therefore, it is maybe interesting to look to more primitive species like insects, invertebrates and plants, which do not have a specialized adaptive immune system to protect them. Is the surface of these organisms only a passive mechanical barrier?

Animals and plants came late to a world that was already populated by microorganisms. Primitive species do not have the protection of professional immune cells. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes and antibodies. A plant seed germinates successfully in the midst of soil microbes. Consequently, the development of potent and broadly effective host defense mechanisms was essential to their survival. Luckily nature has seen to that. All species throughout the plant and animal kingdom use antimicrobial peptides to protect their multicellular organisms. Despite their ancient lineage, antimicrobial peptides have remained effective defensive weapons using the negatively charged outermost leaflet of the bacterial membrane bilayer as an 'Achilles heel'. Animals and plants have cell membranes composed principally of lipids with no net charge (most of the lipids with negatively charged headgroups are segregated into the inner leaflet, facing the cytoplasm), and are not threatened by these natural cationic peptides.

The antimicrobial peptides are believed to kill microorganisms via channel or pore formation. The presence of small 'cationic cysteine-rich' antimicrobial proteins in mammals was first reported in the 1960s but it took almost 20 years before the first group of peptides, the a-defensins were sequenced and localized in granulocytes and human neutrophils. Ten years ago b-defensins were discovered in bovine granulocytes and tracheal epithelial cells and since then b-defensins have been the subject of elaborate research. These defensins display broad spectrum antimicrobial activity under low salt conditions (physiological conditions) and are gene-encoded. They are constitutively or inducibly expressed in human epithelia and mucosal surfaces and have the ability to kill both gram-positive and gram-negative bacteria, fungi, eukaryotic parasites and enveloped viruses.

In their strategy to act as agents of host defense, b-defensins (1) target microbial structures essential for survival and not readily altered by simple genetic changes, (2) use a combination of agents to increase effectiveness (overlapping spectra or synergy between different antimicrobial peptides), (3) act as multifunctional agents by modulating inflammatory response, wound repair, cell division and the adaptive immune response. By acting as signaling molecules, some defensins can increase protection against microbial infection by activating other host defenses. This function could explain their biological effects when their concentrations are too low to be directly microbicidal. Together, the function of these defensins expressed and produced on mucosal surfaces can be seen as a combined host defense system by linking direct bactericidal activity with recruitment of inflammatory cells and wound healing. But is this defense regulated? Do the antigens that encounter the mucosal surfaces influence these antimicrobial peptides. And if there is an antigen–mucosal interaction, how does it work?

Examination of the intracellular pathway involved in the regulation of antimicrobial peptides revealed that in Drosophila this pathway is initiated by signaling through a receptor called Toll. These Toll receptors can bind directly with LPS and other microbial products. Their fast recognition of foreign antigenic matter is based on the recognition of pathogen associated molecular patterns (PAMP's) as non-self. Currently, the mammalian TLR family consists of 10 members (TLR 1 to TLR 10) and the members of this family are responsible for recognizing microbial products: LPS, peptoglycan, bacterial CpG DNA motifs... The expression pattern of each TLR varies but is usually associated with cells of the innate immune system: macrophages, dendritic cells (DC), endothelial cells and mucosal epithelial cells. After binding with extracellular PAMP's, the intracellular domain of the TLR activates a signaling pathway resulting in NF-kB activation leading to expression of genes important for innate and adaptive immunity. Chemokines, cytokines, adhesion molecules, iNOS, and antimicrobial peptides, which all contribute to the early host response against the invading pathogen are encoded by these genes. But also the adaptive immunity is influenced by this TLR triggering. The abovementioned signaling molecules directly influence adaptive responses, but also co-stimulatory molecules such as CD40, CD80 and CD86 found on DC and activated B cells and macrophages are up-regulated by LPS and PGN, showing an adaptive response to the microbial challenge.

The mucosae of ear, nose, and throat provide not only a barrier against invading microorganisms; it is also a site of active interaction with the innate and adaptive immune system. It is well known that the adaptive immunity obtains its information from the important mucosal associated lymphoid tissue like tonsils and adenoids so it can mature and be standby with a clonal defense mechanism. At the other side we have a very large barrier surface that is also capable of sending information about the outside micro-organical matter to the immune system without destruction of this barrier or the induction of disease/inflammation. The epithelium has a crucial role in the quick and quite innate defense against foreign matter not only by a rigid barrier function but with powerful, flexible and specific tools.

The recently discovered Toll like receptors are possible candidates to play a crucial role in this mucosal–antigen interaction.