Respiration 1992;59(suppl 3):3—13
Departments of Otolaryngology and Pediatrics. State University of New York at Buffalo, and Division of Infectious Diseases. Buffalo Children's Hospital. Buffalo. N.Y.. USA
Mucosal Immunology of the Upper Respiratory Tract
Key Words
Abstract
Bacterial interference Mucosal immunity Nasal mucosa Tonsils and adenoids
The palatine tonsils anti nasopharyngeal adenoids represent the predominant immunocompetent tissue of the upper respiratory tract. Its major function is as a first line of defense against viral, bacterial, and food antigens that enter the upper aerodigestive system. Another major function of the tonsils and adenoids is to supply the local mucosal immune system of the upper respiratory tract with dimeric IgA-producing B cells. Secretory IgA has particular hydrophilic proper ties and is capable of preventing adsorption and penetration of bacteria and/or viruses into the upper respiratory tract mucosa. In addition, the role of the indi genous flora of the upper respiratory tract, particularly the viridans streptococci has been emphasized as providing a valuable source of bacterial interference to the colonization of potential pathogens.
Introduction
Inflammatory diseases of the upper respiratory tract caused by viruses and bacteria constitute the most common cause of primary care visits to pediatricians and primary care physicians. Despite the availability of multiple anti biotics and vaccinations for many serious viral and bacterial diseases that involve the upper respiratory tract, physicians are challenged today by the overwhelming increase in otitis media and sinusitis caused by the ever-increasing resistance of bacteria to commonly used chemotherapeutic agents. Some of these bacteria, such as nontypable Haemophilus influenzae (NTHI) have been considered normal commen sals of the upper respiratory tract [ 1]. It is obvious then that new strategies will be required to control upper respiratory tract infections. The knowledge for combating this problem requires a thorough understanding of two basically differ ent mechanisms which appear to control the population size that a given bacterial species or strain can achieve on mucosal surfaces, namely: (1) the local immune mech
anisms. and (2) the antagonistic action of other bacterial species comprising the normal bacterial flora [2], The two mechanisms may be functionally interdependent. The purpose of this review is to briefly outline the secretory immune system of the upper respiratory tract mucosa and to resurrect an old but very important concept: the role of the normal indigenous flora in bacterial interference.
Upper Respiratory Tract Immunology
Mucosal immunology of the upper respiratory tract requires a thorough understanding of the sources of immu nocompetent cells that populate the mucus membrane of the upper respiratory tract. The lymphoid tissue of the Waldeyer ring, comprising the palatine tonsils, the naso pharyngeal tonsils (adenoids), and the lingual tonsils repre sent probably the most important lymphoepithelial tissue of the upper respiratory tract. Recent evidence suggests
Joel M. Bernstein. MD. PhO Departments of Otolaryngology and Pediatrics. State University of Now York at Buffalo and the Division of Infectious Diseases. Buffalo Children’s Hospital 219 Bryant Street. Buffalo. N.Y. (USA)
© 1992 Kargcr A G. Basel 0025-7931/92/0599-0003 S 2.75/0
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J.M. Bernstein
that in the normal healthy adult, at least, the bronchopul monary lymphoid tissue, so-called BALT, does not repre sent an important precursor of immunocompetent cells for the upper respiratory tract [3], Therefore, primary empha sis in this review will be on the tonsils and adenoids. In addi tion, the nasal mucosa represents also a first line of defense against inhaled, viral, bacterial and other foreign antigens, and will also be thoroughly discussed.
Bernstein
Table 1. Distribution of T cells and T cell subsets in blood, tonsils and adenoids
CD3 (total T cells) CD4 T,,,,, CDS T(S(,
Blood
Tonsil
Adenoid
66 ±8 35 ±7 29 ±6
42 ±2 32 ±10 8±3
41 ±5 30+7 7±2
Table 2. Distribution of immunoglobulin-bearing cells (B cells)
Blood (n=46)
IgD IgM IgA IgG
Tonsil
Adenoid (n = 9)
total
%
total
%
total
%
2.5 ±0.33 9.8 ±0.82 0.32 ±0.10 2.1 ±0.42
22 77 4.6 18
1.2+0.18 19.9 ± 1.4 7.4 ±0.53 31.7 ±2.1
8 43 20 74
1.8 ±58 22.9 ±3.9 5.8± 1.2 37.7 ±2.5
4 46 14 74
Table 3. Distribution of T cells and T cell subsets in blood, tonsils and adenoids
CD3 l.eu 8+ (SI) CD3 Leu 8- (HI)
Blood
Tonsil
Adenoid
29 ±9 7±7
I0±8 21 ± 9
9±7 21 ±5
Mucosal Immunology
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4
Fig. 1. High-power photomicrograph of the morphology of the tonsil surrounding a crypt (C). Antigen enters the crypt and is taken up by cells of the reticulum epithelium (RE). Antigen-presenting cells in this region consist of Langerhans cells, macrophages and perhaps M cells. These cells then transport antigen to T cells in the extrafollicular zone (EZ). In addition, antigen presentation to cells in the mantle zone (MZ.) may stimulate memory cells with surface IgD and IgM to switch to more mature clones of B cells possessing IgA and IgG. Finally, specific dendritic cells in the germinal center (GC) may be responsible for antigen presentation to B cells in this area.
II c
Immunobiology o f the Tonsils and Adenoids The palatine tonsils and nasopharyngeal tonsils have a characteristic lymphoid architecture which includes a retic ular epithelium, a follicular region, or primary and second ary germinal centers, which are covered with mantle zones and extrafollicular regions [4], The immunocompetent cells that arc present in these various regions of the tonsil have been thoroughly studied by many investigators using spe cific polyclonal and monoclonal antibodies directed against specific epitopes of lymphocytes. Figure 1 summarizes the immunocytoarchitecture of the palatine tonsil. The princi pal route of antigen uptake occurs in the crypts of the pala tine tonsils and the furrows of the nasopharyngeal tonsil. In the human palatine tonsil, there are 10-20 crypts, which tremendously increases the exposure for antigen uptake. There are cells which may be important in antigen uptake so-called M cells, which are also present in Peyer's patches of the small bowel [5]. In addition, there are many HLADR-positive cells deep in the crypt which may also be important in antigen uptake. However, it is likely that the most important cells that arc involved in antigen transport are macrophages, interdigitating cells, which are present in the extrafollicular areas, and follicular dendritic cells, which are primarily present in the germinal center |6], Thymus-derived, or T cells, characteristically occupy the interfollicular zones of the palatine and nasopharyngeal tonsils, but the helper-inducer T lymphocyte is also present in significant quantities in the germinal centers [7]. B cells are primarily present in the germinal center and mantle zone, but may also be found in the reticular epithelium. Using the fiuorescinated-activated cell sorter (FACS), we have measured the percentage of various T cell subsets in the palatine tonsils and adenoids. In addition, the percent age of different types of B cells has also been calculated. The data are summarized in tables 1-3. Whereas T cells represent the majority of lymphocytes in the peripheral blood. B cells predominate in the tonsils and adenoids. Fur thermore, the ratio between helper-inducer and suppressor cytotoxic cells is slightly above 1 in the peripheral blood. The ratio is significantly higher in the tonsils and adenoids (table 1). The distribution of various immunoglobulin-
Fig. 2. Immunological histological localization of IgA using FITC-conjugatcd anti-immunoglobulin A antibody. IgA plasma cells are found in the lamina propria underlying the crvpi (Cr) epithelium and in the intcrfollicular area.
Fig. 3. Immunological localization of IgG immunocytes in the tonsillar tissue. There arc multiple immunocytes possessing IgG in the interfollicular area. There is virtually no IgG in the mantle zone (mz) and lacy staining between the cells is seen in the germinal center (gc). cc = Crypt epithelium.
to areas of the upper respiratory tract mucosa. This brief summary of the immunological cytoarchitecture of the palatine and nasopharyngeal tonsils suggests that these lymphoepithelial structures possess all of the elements required for a local immune system. Bacterial, viral, or food antigen can be selectively absorbed by macrophages, HLA-positive cells. M cells, or interdigitating cells in these lymphoepithelial tissues. Furthermore, the antigen can be transported in this way to both T cells in the extrafollicular Downloaded by: Stockholm University Library 130.237.165.40 - 4/24/2018 6:07:15 PM
bearing B cells is summarized in table 2. In the peripheral blood, IgM represents the most common B cells, whereas in the tonsils. IgG is the most common cell found. The dis tribution of immunoglobulin-bearing cells in the tonsils and adenoids is almost identical (table 2). Further subclassification of T cells is summarized in table 3. The C D3+ Lcu8+ cells have been found to be suppressor-inducer cells, whereas the CD3+ Leu8- cells have been found to be helper-inducer cells |8|. There is a signi ficant difference between the distribution of the suppressorinducer and helper-inducer cells between the blood and lymphoepithelial tissue. The helper-inducer cell is more common in the tonsil, whereas the suppressor-inducer cell is found more frequently in the peripheral blood (table 3). Using immunohistological localization of immunoglobins, various investigators have studied the palatine tonsils [9. 10]. The immunocyte, or plasma cell, comprises no more than 2% of the total cells in the palatine tonsil, and some what less in the nasopharyngeal tonsil 111]. IgG appears to be the predominant immunocyte and the relationship between IgA and IgM differs according to the methods of study used. Studying cytoplasmic staining of immunocytes. Brandtzaeg et al. 112] have suggested that IgA immuno cytes arc significantly greater than IgM immunocytes. However, using the ELISPOT technique, which specif ically identifies cells that are actively secreting immunoglo bulin. our laboratory has found that the IgA- and IgMsecrcting cells appear to be similar in frequency [ 111. How ever. IgA secretion in the palatine tonsils is significantly greater than in the lymph nodes [ 13]. suggesting that the palatine and nasopharyngeal tonsils have characteristics of a local immune system. The mantle zones of the tonsil appear to be very specific for early clones of B cells elaborating both IgM and IgD on their surfaces |7], In the germinal center. IgG and IgM are most frequently found, suggesting that there is a significant switching of early clones of B cells to more mature B cells in the germinal center [14|. Finally. IgA and IgG immuno cytes are frequently found in the crypt epithelium as well as the extrafollicular zones (fig. 2, 3). A summary of the distri bution of different immunocompetent cells in the tonsils in different age groups using image analysis is shown in figures 4 and 5. The production of .1 chain by different B cells in the palatine tonsils has been a particularly important contribu tion from the Brandtzaeg laboratory and represents a very important concept in regard to the relationship between normal and diseased tonsils [15]. It also is an important con cept in regard to the putative function of the tonsil as a pre cursor of early clones of B cells which are J-chain-positive
6
Bernstein
Fig. 4. A summary of the distribution of immunocompetent cells (T cell subsets and B cell subsets) in the normal tonsil with the use of image analysis with monoclonal antibodies as adapted from N. Yamanaka et al. [7]. IgD and IgM predominate in the mantle zone (MZ). whereas IgM and IgG predominate in the germinal center (GC). IgG is the predominant immunoglobulin in both the reticular epithelium and the interfollicular areas. The image analysis technique takes into account both immunocytes as well as B cells.
Fig. 5. The distribution of immunocompetent cells in the aged tonsil shows that most immunoglobulins have decreased significantly in all compartments of the tonsil.
Mucosal Immunology
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area by interdigitating cells and by follicular dendritic cells to B cells in the germinal center. Early clones of B cells in the mantle zone possessing surface IgM and IgD may be memory cells which can be stimulated into more mature clones of B cells. With the proper stimulation of B cells with both early and late interleukins from T-helper cells in the follicular zone. B cells may mature into either memory cells or into immunoglobulin-synthesizing plasma cells [16]. These plasma cells are then distributed in the extrafollicular zone and crypt epithelium, and immunoglobulins will then be secreted into the crypt. In this way, the tonsil plays an important role in maintaining the normal microbiological flora in the crypts of the tonsils and adenoids, at least in the normal healthy person, and finally, B cells which possess J chains will mature into IgM and IgA plasma cells, which will secrete J-chain-positive immunoglobulins. The naso pharyngeal tonsil which possesses a secretory component will function as a true secretory immune system, whereas the palatine tonsil which possesses either stratified squa mous epithelium or simple cuboidal epithelium as in the crypt, will secrete IgA into the crypt lumen, but may also be the source of J-chain-positive IgA B cells for other areas of the upper respiratory system, such as the parotid gland, lacrimal gland, nasal mucosa and middle ear mucosa in otitis media (fig.6). All secretory sites of adults normally contain a remarkable predominance of IgA-producing immunocytes. A relatively large proportion of IgA: has been reported for the IgA immunocytes present in secre tory sites in the colon [ 18], whereas the tonsils possess pri marily IgA, immunocytes, suggesting that there is a signi ficant dichotomy of the secretory immune system and that the source of IgA B cells may be different for the upper respiratory tract and lower gastrointestinal tract (table 4). In addition to the above-mentioned immunocompetent cells and antigen-presenting cells, it has been recently shown that the tonsil also possesses all of the necessary molecules that play a crucial role in adhesion and signal transduction 119. 20]. Vitronectin, fibronectin. laminin, as well as other surface-associated adhesion molecules arc present in tonsillar lymphocytes. The microvasculature of the tonsil also possesses lymphocyte-associated antigen l (LFA-1), as well as intercellular adhesion molecule-1 (ICAM -l). which is expressed on the endothelium of the microvasculature of the tonsil. This expression of LFA-1 on a large number of tonsillar lymphocytes, together with the up-regulated expression of ICA M -l. suggests that the high endothelial venules (HEV) of the palatine tonsils occupy a strategic position in promoting lymphocyte extravasation into this tissue. Inasmuch as normal human lungs lack organized bronchus-associated lymphoid tissue, which is
Tonsil
Adenoid
Secondary Signals T cells — Antigens Hormones? S aliva^ Glands
Nasal Mucosa
Lacrimal Gland
s
Middle Ear
IgA
1.
Prevents Adsorption ot Viruses and Bacteha to Epithelial Surfaces by Increasing Hydrophilicity ol Organism.
2.
Act as Blocking Antibody (Anti-Inflammatory Immunoglobulins)
prominent in certain animal species, the major source of B cell precursors of the upper respiratory tract of humans appears to be the lymphoid tissue of Waldeyer’s ring domi nated by the palatine tonsil. Mucosal Immunology o f the Nose The mucosa that lines the nasal cavity is protected by a secretory immune system which is under complex regula tory control. As mentioned in a previous section, B cells of relatively immature memory clones with a potential for J chain expression are initially stimulated in the tonsil and adenoid and migrate to glandular sites where they arc sub jected to terminal differentiation and then become immu noglobulin-producing immunocytcs. The secretory IgA system as part of the mechanism of immune exclusion is of paramount importance in this regard and its physicochemi cal properties and its ability to prevent adsorption of bacte ria or viral particles to the epithelial surfaces of the nose should be briefly emphasized. It has been observed that secretory IgA reduces the negative surface charge and lia bility to hydrophobic interaction and decreases the associ ation of phagocytosis-sensitive bacteria with polymorphnuclear leukocytes [21]. By contrast, hyperimmune IgG
(SIgA), which is the major immunoglobulin that results in immune exclusion of virus particles, bacteria, and perhaps food antigens. The mechanism of SIgA involves the enhancement of hydrophilicity of the surface of bacterial organisms or viral particles. Also. SIgA may block the activity of IgG. and in this way. may function as an anti-inflamma tory immunoglobulin.
Table 4. Dichotomy of the secretory immune system
Lymphoepithelial organ
MALT
Immuno globulins
T switching
Tonsil, adenoid
nasal mucosa
IgA, IgD > IgM
Peyer’s patches
intestinal mucosa
IgA; IgM>IgD
sequential from IgM - IgD - IgG -» IgA, or IgD — IgA, IgM —» IgA;
MALT = Mucosa associated lymphoid tissue
raised against virulent strains of bacteria produce a hydrophobic effect and promote phagocytosis in vivo as well as in vitro [22]. The pioneering work of van Oss in Buffalo has demonstrated that both IgA, and especially secretory' IgA, increases the hydrophilicity of the bacterial surface, and thus makes it less available for opsonization and engulfment by neutrophils [23]. Therefore, the mucin layer of the nasal mucosa acts as a mechanical barrier against the penetration of particulate
7
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Fig. 6. Summary of the mucosal immunity of the upper respira tory tract. The tonsils and the adenoids possess early and late clones of B cells that migrate to other areas of the upper respiratory' tract, including the salivary’ glands, the lacrimal gland, the middle ear and the nasal mucosa. J-chain-positive IgA cells that migrate to these areas may he stimulated by secondary’ signals, such as specific antigens. T cells, or hormones. This will lead to the production of secretory IgA
8
Bernstein
lilliL .
Epithelium
•
•
Sub-epithelium
ú
•
J iliH . » •
IgG IgA IgM
(50) (50) (1)
igo
(1)
•
•
CD8 B cells CD4 (57) CD8 (22) B cells (21) CD4 (39) CD8 (50) B cells (11)
Glandular area IgM (2) IgD (2)
IgA (80) IgG (20)
@
Deep Vascular area
CD4 (57 CD8 (30 B cells (12
Fig. 7. I his diagram summarizes the distribution of immunocom petent cells (%) in the nasal mucosa [adapted from W intheret at.. 27. and Brandtzacg. 24], There is a preponderance of T helper cells in the epithelium: however, in the intcrglandular area, the CD8 cells, or cytotoxic suppressor cells, predominante. The distribution of immu noglobulins. according to Brandtzacg [24] is also summarized. Whereas in the subepilhelium the distribution for IgG and IgA is approximately equal, there is a marked predominance of IgA immu nocytes in the intcrglandular area.
in about 70% of IgG immunocytes and in almost 100% of IgD immunocytes [24], The Nasal Mucosa as an Immunocompetent Tissue Another immunological function of the nasal mucosa may be related to the production of local antibody to incoming inhaled antigen. As mentioned above, it is likely that most antigens are prevented from entering into the nasal mucosa by so-called ‘immune exclusion'. Specifically, such organisms as NTH I and Streptococcus pneumoniae are inhibited from adhering to both the nasopharyngeal and the nasal mucosas [25]. Specific IgA present in external may play an important role in the pathogenesis of otitis media in children. In general, all of the immunocompetent cells necessary for the induction of specific antibody are present in the nasal mucosa. The distribution and number of lympho cytes, monocytes, and cells expressing I1LA-DR antigen have been studied in a number of investigations in healthy adults using monoclonal antibody avidin-biotin immuno peroxidase techniques. A summary of the findings of Whi ther et al. [27] and Brandtzacg |24] is graphically summa rized in figure 7. In general, T helper cells appear to be more predominant in the superficial layers of the muco
Mucosa! Immunology
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and molecular matter, not only by the mechanical cleaning of the mucociliary system, but by the local immune mechanism of secretory IgA. In addition to secretory IgA. there arc other nonspecific mechanisms that the nasal mucosa has available to it. including the release of lactoferrin and lysozyme [24]. Lactoferrin is an iron-binding pro tein which exerts a bactericidal and bacteriostatic effect on both gram-positive and gram-negative bacteria, as well as Candida. Lactoferrin also appears to be enhanced by secre tory IgA. Lysozyme, or muramidase. is also bactericidal and bacteriostatic for certain gram-positive bacteria and accelerates the lytic effect of antibody-activated comple ment on certain gram-negative bacteria which may also involve secretory IgA. Both lactoferrin and lysozyme seem to be selectively produced by the serous glandular element and are absent from the mucus glandular elements of the nasal mucosas. In addition to secretory IgA. which is the major immu noglobulin that is active in immune exclusion, other immu noglobulin isotypes are present in the nasal mucosa. IgG is present primarily in the lamina propria, whereas secretory IgA is primarily present around the glandular elements deeper in the mucosa [24]. However, there is a great deal of IgG which is present in the nasal mucosas as a transudate from the serum. It has been demonstrated by a number of investigators that in inflammatory rhinitis, there is an increase in the number of IgG-producing immunocytes and a decrease in the number of IgA immunocytes [24], In nor mal nasal mucosa, Brandzaeg [24] has shown that 76.7% of IgA is gland-associated, whereas 46% is surface-associ ated. whereas in inflammatory rhinitis, approximately 62% of IgA is gland-associated and 24% is surface-associated. In addition to secretory IgA and IgG. the nasal mucosa pos sesses many more IgD immunocytes than any other secre tory mucosal surface in the body, whereas in the gastro intestinal tract. lgM appears to be more predominant. IgEproducing immunocytes are virtually absent from normal nasal mucosa but are apparently somewhat increased in allergic nasal mucosa. There is still a significant controversy as to whether most of the IgE present in the nasal mucosa is associated with mucosal and connective tissue mast cells rather than IgE-producing immunocytes. In summary, the glandular areas of the nasal mucosa represent an extremely potent site of IgA formation, whereas little IgM is produced locally. On the other hand, compared with normal intestinal mucosa and exocrine glands, a relatively large amount of IgG is produced in the mucosa even in the absence of inflammation. A remarkable number of IgA and IgM cells in the nasal mucosa arc J-chain-positive. J chain is present in free form
32
16 Antibody Titer 8 ya
•
•
••
•
tm
mi
— •—
— •—
—
•
•
• m
4 2
T Live Vaccine
18
1 22
26
• 30
1 34
Months
Fig. 8. Levels of SIgA poliovirus type I antibody in the nasopha rynx after primary immunization with trivalent live attenuated polio virus vaccine in healthy children whose tonsils have not been removed. The individual and mean antibody titers are shown. The antibody titers maintain their levels for a period of at least 34 months.
Fig. 9. Level of IgA poliovirus antibody in the nasopharynx before and after tonsillectomy and adenoidectomy (T&A). The chil dren were immunized with live polio vaccine 6 months to 6 years before the operation. Individual and mean geometric mean titers arc shown. The mean antibody titers decrease three-fold after the removal of tonsils and adenoids from 11.8 before and 3.9 after the operation (p
Departments of Otolaryngology and Pediatrics. State University of New York at Buffalo, and Division of Infectious Diseases. Buffalo Children's Hospital. Buffalo. N.Y.. USA
Mucosal Immunology of the Upper Respiratory Tract
Key Words
Abstract
Bacterial interference Mucosal immunity Nasal mucosa Tonsils and adenoids
The palatine tonsils anti nasopharyngeal adenoids represent the predominant immunocompetent tissue of the upper respiratory tract. Its major function is as a first line of defense against viral, bacterial, and food antigens that enter the upper aerodigestive system. Another major function of the tonsils and adenoids is to supply the local mucosal immune system of the upper respiratory tract with dimeric IgA-producing B cells. Secretory IgA has particular hydrophilic proper ties and is capable of preventing adsorption and penetration of bacteria and/or viruses into the upper respiratory tract mucosa. In addition, the role of the indi genous flora of the upper respiratory tract, particularly the viridans streptococci has been emphasized as providing a valuable source of bacterial interference to the colonization of potential pathogens.
Introduction
Inflammatory diseases of the upper respiratory tract caused by viruses and bacteria constitute the most common cause of primary care visits to pediatricians and primary care physicians. Despite the availability of multiple anti biotics and vaccinations for many serious viral and bacterial diseases that involve the upper respiratory tract, physicians are challenged today by the overwhelming increase in otitis media and sinusitis caused by the ever-increasing resistance of bacteria to commonly used chemotherapeutic agents. Some of these bacteria, such as nontypable Haemophilus influenzae (NTHI) have been considered normal commen sals of the upper respiratory tract [ 1]. It is obvious then that new strategies will be required to control upper respiratory tract infections. The knowledge for combating this problem requires a thorough understanding of two basically differ ent mechanisms which appear to control the population size that a given bacterial species or strain can achieve on mucosal surfaces, namely: (1) the local immune mech
anisms. and (2) the antagonistic action of other bacterial species comprising the normal bacterial flora [2], The two mechanisms may be functionally interdependent. The purpose of this review is to briefly outline the secretory immune system of the upper respiratory tract mucosa and to resurrect an old but very important concept: the role of the normal indigenous flora in bacterial interference.
Upper Respiratory Tract Immunology
Mucosal immunology of the upper respiratory tract requires a thorough understanding of the sources of immu nocompetent cells that populate the mucus membrane of the upper respiratory tract. The lymphoid tissue of the Waldeyer ring, comprising the palatine tonsils, the naso pharyngeal tonsils (adenoids), and the lingual tonsils repre sent probably the most important lymphoepithelial tissue of the upper respiratory tract. Recent evidence suggests
Joel M. Bernstein. MD. PhO Departments of Otolaryngology and Pediatrics. State University of Now York at Buffalo and the Division of Infectious Diseases. Buffalo Children’s Hospital 219 Bryant Street. Buffalo. N.Y. (USA)
© 1992 Kargcr A G. Basel 0025-7931/92/0599-0003 S 2.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 4/24/2018 6:07:15 PM
J.M. Bernstein
that in the normal healthy adult, at least, the bronchopul monary lymphoid tissue, so-called BALT, does not repre sent an important precursor of immunocompetent cells for the upper respiratory tract [3], Therefore, primary empha sis in this review will be on the tonsils and adenoids. In addi tion, the nasal mucosa represents also a first line of defense against inhaled, viral, bacterial and other foreign antigens, and will also be thoroughly discussed.
Bernstein
Table 1. Distribution of T cells and T cell subsets in blood, tonsils and adenoids
CD3 (total T cells) CD4 T,,,,, CDS T(S(,
Blood
Tonsil
Adenoid
66 ±8 35 ±7 29 ±6
42 ±2 32 ±10 8±3
41 ±5 30+7 7±2
Table 2. Distribution of immunoglobulin-bearing cells (B cells)
Blood (n=46)
IgD IgM IgA IgG
Tonsil
Adenoid (n = 9)
total
%
total
%
total
%
2.5 ±0.33 9.8 ±0.82 0.32 ±0.10 2.1 ±0.42
22 77 4.6 18
1.2+0.18 19.9 ± 1.4 7.4 ±0.53 31.7 ±2.1
8 43 20 74
1.8 ±58 22.9 ±3.9 5.8± 1.2 37.7 ±2.5
4 46 14 74
Table 3. Distribution of T cells and T cell subsets in blood, tonsils and adenoids
CD3 l.eu 8+ (SI) CD3 Leu 8- (HI)
Blood
Tonsil
Adenoid
29 ±9 7±7
I0±8 21 ± 9
9±7 21 ±5
Mucosal Immunology
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4
Fig. 1. High-power photomicrograph of the morphology of the tonsil surrounding a crypt (C). Antigen enters the crypt and is taken up by cells of the reticulum epithelium (RE). Antigen-presenting cells in this region consist of Langerhans cells, macrophages and perhaps M cells. These cells then transport antigen to T cells in the extrafollicular zone (EZ). In addition, antigen presentation to cells in the mantle zone (MZ.) may stimulate memory cells with surface IgD and IgM to switch to more mature clones of B cells possessing IgA and IgG. Finally, specific dendritic cells in the germinal center (GC) may be responsible for antigen presentation to B cells in this area.
II c
Immunobiology o f the Tonsils and Adenoids The palatine tonsils and nasopharyngeal tonsils have a characteristic lymphoid architecture which includes a retic ular epithelium, a follicular region, or primary and second ary germinal centers, which are covered with mantle zones and extrafollicular regions [4], The immunocompetent cells that arc present in these various regions of the tonsil have been thoroughly studied by many investigators using spe cific polyclonal and monoclonal antibodies directed against specific epitopes of lymphocytes. Figure 1 summarizes the immunocytoarchitecture of the palatine tonsil. The princi pal route of antigen uptake occurs in the crypts of the pala tine tonsils and the furrows of the nasopharyngeal tonsil. In the human palatine tonsil, there are 10-20 crypts, which tremendously increases the exposure for antigen uptake. There are cells which may be important in antigen uptake so-called M cells, which are also present in Peyer's patches of the small bowel [5]. In addition, there are many HLADR-positive cells deep in the crypt which may also be important in antigen uptake. However, it is likely that the most important cells that arc involved in antigen transport are macrophages, interdigitating cells, which are present in the extrafollicular areas, and follicular dendritic cells, which are primarily present in the germinal center |6], Thymus-derived, or T cells, characteristically occupy the interfollicular zones of the palatine and nasopharyngeal tonsils, but the helper-inducer T lymphocyte is also present in significant quantities in the germinal centers [7]. B cells are primarily present in the germinal center and mantle zone, but may also be found in the reticular epithelium. Using the fiuorescinated-activated cell sorter (FACS), we have measured the percentage of various T cell subsets in the palatine tonsils and adenoids. In addition, the percent age of different types of B cells has also been calculated. The data are summarized in tables 1-3. Whereas T cells represent the majority of lymphocytes in the peripheral blood. B cells predominate in the tonsils and adenoids. Fur thermore, the ratio between helper-inducer and suppressor cytotoxic cells is slightly above 1 in the peripheral blood. The ratio is significantly higher in the tonsils and adenoids (table 1). The distribution of various immunoglobulin-
Fig. 2. Immunological histological localization of IgA using FITC-conjugatcd anti-immunoglobulin A antibody. IgA plasma cells are found in the lamina propria underlying the crvpi (Cr) epithelium and in the intcrfollicular area.
Fig. 3. Immunological localization of IgG immunocytes in the tonsillar tissue. There arc multiple immunocytes possessing IgG in the interfollicular area. There is virtually no IgG in the mantle zone (mz) and lacy staining between the cells is seen in the germinal center (gc). cc = Crypt epithelium.
to areas of the upper respiratory tract mucosa. This brief summary of the immunological cytoarchitecture of the palatine and nasopharyngeal tonsils suggests that these lymphoepithelial structures possess all of the elements required for a local immune system. Bacterial, viral, or food antigen can be selectively absorbed by macrophages, HLA-positive cells. M cells, or interdigitating cells in these lymphoepithelial tissues. Furthermore, the antigen can be transported in this way to both T cells in the extrafollicular Downloaded by: Stockholm University Library 130.237.165.40 - 4/24/2018 6:07:15 PM
bearing B cells is summarized in table 2. In the peripheral blood, IgM represents the most common B cells, whereas in the tonsils. IgG is the most common cell found. The dis tribution of immunoglobulin-bearing cells in the tonsils and adenoids is almost identical (table 2). Further subclassification of T cells is summarized in table 3. The C D3+ Lcu8+ cells have been found to be suppressor-inducer cells, whereas the CD3+ Leu8- cells have been found to be helper-inducer cells |8|. There is a signi ficant difference between the distribution of the suppressorinducer and helper-inducer cells between the blood and lymphoepithelial tissue. The helper-inducer cell is more common in the tonsil, whereas the suppressor-inducer cell is found more frequently in the peripheral blood (table 3). Using immunohistological localization of immunoglobins, various investigators have studied the palatine tonsils [9. 10]. The immunocyte, or plasma cell, comprises no more than 2% of the total cells in the palatine tonsil, and some what less in the nasopharyngeal tonsil 111]. IgG appears to be the predominant immunocyte and the relationship between IgA and IgM differs according to the methods of study used. Studying cytoplasmic staining of immunocytes. Brandtzaeg et al. 112] have suggested that IgA immuno cytes arc significantly greater than IgM immunocytes. However, using the ELISPOT technique, which specif ically identifies cells that are actively secreting immunoglo bulin. our laboratory has found that the IgA- and IgMsecrcting cells appear to be similar in frequency [ 111. How ever. IgA secretion in the palatine tonsils is significantly greater than in the lymph nodes [ 13]. suggesting that the palatine and nasopharyngeal tonsils have characteristics of a local immune system. The mantle zones of the tonsil appear to be very specific for early clones of B cells elaborating both IgM and IgD on their surfaces |7], In the germinal center. IgG and IgM are most frequently found, suggesting that there is a significant switching of early clones of B cells to more mature B cells in the germinal center [14|. Finally. IgA and IgG immuno cytes are frequently found in the crypt epithelium as well as the extrafollicular zones (fig. 2, 3). A summary of the distri bution of different immunocompetent cells in the tonsils in different age groups using image analysis is shown in figures 4 and 5. The production of .1 chain by different B cells in the palatine tonsils has been a particularly important contribu tion from the Brandtzaeg laboratory and represents a very important concept in regard to the relationship between normal and diseased tonsils [15]. It also is an important con cept in regard to the putative function of the tonsil as a pre cursor of early clones of B cells which are J-chain-positive
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Fig. 4. A summary of the distribution of immunocompetent cells (T cell subsets and B cell subsets) in the normal tonsil with the use of image analysis with monoclonal antibodies as adapted from N. Yamanaka et al. [7]. IgD and IgM predominate in the mantle zone (MZ). whereas IgM and IgG predominate in the germinal center (GC). IgG is the predominant immunoglobulin in both the reticular epithelium and the interfollicular areas. The image analysis technique takes into account both immunocytes as well as B cells.
Fig. 5. The distribution of immunocompetent cells in the aged tonsil shows that most immunoglobulins have decreased significantly in all compartments of the tonsil.
Mucosal Immunology
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area by interdigitating cells and by follicular dendritic cells to B cells in the germinal center. Early clones of B cells in the mantle zone possessing surface IgM and IgD may be memory cells which can be stimulated into more mature clones of B cells. With the proper stimulation of B cells with both early and late interleukins from T-helper cells in the follicular zone. B cells may mature into either memory cells or into immunoglobulin-synthesizing plasma cells [16]. These plasma cells are then distributed in the extrafollicular zone and crypt epithelium, and immunoglobulins will then be secreted into the crypt. In this way, the tonsil plays an important role in maintaining the normal microbiological flora in the crypts of the tonsils and adenoids, at least in the normal healthy person, and finally, B cells which possess J chains will mature into IgM and IgA plasma cells, which will secrete J-chain-positive immunoglobulins. The naso pharyngeal tonsil which possesses a secretory component will function as a true secretory immune system, whereas the palatine tonsil which possesses either stratified squa mous epithelium or simple cuboidal epithelium as in the crypt, will secrete IgA into the crypt lumen, but may also be the source of J-chain-positive IgA B cells for other areas of the upper respiratory system, such as the parotid gland, lacrimal gland, nasal mucosa and middle ear mucosa in otitis media (fig.6). All secretory sites of adults normally contain a remarkable predominance of IgA-producing immunocytes. A relatively large proportion of IgA: has been reported for the IgA immunocytes present in secre tory sites in the colon [ 18], whereas the tonsils possess pri marily IgA, immunocytes, suggesting that there is a signi ficant dichotomy of the secretory immune system and that the source of IgA B cells may be different for the upper respiratory tract and lower gastrointestinal tract (table 4). In addition to the above-mentioned immunocompetent cells and antigen-presenting cells, it has been recently shown that the tonsil also possesses all of the necessary molecules that play a crucial role in adhesion and signal transduction 119. 20]. Vitronectin, fibronectin. laminin, as well as other surface-associated adhesion molecules arc present in tonsillar lymphocytes. The microvasculature of the tonsil also possesses lymphocyte-associated antigen l (LFA-1), as well as intercellular adhesion molecule-1 (ICAM -l). which is expressed on the endothelium of the microvasculature of the tonsil. This expression of LFA-1 on a large number of tonsillar lymphocytes, together with the up-regulated expression of ICA M -l. suggests that the high endothelial venules (HEV) of the palatine tonsils occupy a strategic position in promoting lymphocyte extravasation into this tissue. Inasmuch as normal human lungs lack organized bronchus-associated lymphoid tissue, which is
Tonsil
Adenoid
Secondary Signals T cells — Antigens Hormones? S aliva^ Glands
Nasal Mucosa
Lacrimal Gland
s
Middle Ear
IgA
1.
Prevents Adsorption ot Viruses and Bacteha to Epithelial Surfaces by Increasing Hydrophilicity ol Organism.
2.
Act as Blocking Antibody (Anti-Inflammatory Immunoglobulins)
prominent in certain animal species, the major source of B cell precursors of the upper respiratory tract of humans appears to be the lymphoid tissue of Waldeyer’s ring domi nated by the palatine tonsil. Mucosal Immunology o f the Nose The mucosa that lines the nasal cavity is protected by a secretory immune system which is under complex regula tory control. As mentioned in a previous section, B cells of relatively immature memory clones with a potential for J chain expression are initially stimulated in the tonsil and adenoid and migrate to glandular sites where they arc sub jected to terminal differentiation and then become immu noglobulin-producing immunocytcs. The secretory IgA system as part of the mechanism of immune exclusion is of paramount importance in this regard and its physicochemi cal properties and its ability to prevent adsorption of bacte ria or viral particles to the epithelial surfaces of the nose should be briefly emphasized. It has been observed that secretory IgA reduces the negative surface charge and lia bility to hydrophobic interaction and decreases the associ ation of phagocytosis-sensitive bacteria with polymorphnuclear leukocytes [21]. By contrast, hyperimmune IgG
(SIgA), which is the major immunoglobulin that results in immune exclusion of virus particles, bacteria, and perhaps food antigens. The mechanism of SIgA involves the enhancement of hydrophilicity of the surface of bacterial organisms or viral particles. Also. SIgA may block the activity of IgG. and in this way. may function as an anti-inflamma tory immunoglobulin.
Table 4. Dichotomy of the secretory immune system
Lymphoepithelial organ
MALT
Immuno globulins
T switching
Tonsil, adenoid
nasal mucosa
IgA, IgD > IgM
Peyer’s patches
intestinal mucosa
IgA; IgM>IgD
sequential from IgM - IgD - IgG -» IgA, or IgD — IgA, IgM —» IgA;
MALT = Mucosa associated lymphoid tissue
raised against virulent strains of bacteria produce a hydrophobic effect and promote phagocytosis in vivo as well as in vitro [22]. The pioneering work of van Oss in Buffalo has demonstrated that both IgA, and especially secretory' IgA, increases the hydrophilicity of the bacterial surface, and thus makes it less available for opsonization and engulfment by neutrophils [23]. Therefore, the mucin layer of the nasal mucosa acts as a mechanical barrier against the penetration of particulate
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Fig. 6. Summary of the mucosal immunity of the upper respira tory tract. The tonsils and the adenoids possess early and late clones of B cells that migrate to other areas of the upper respiratory' tract, including the salivary’ glands, the lacrimal gland, the middle ear and the nasal mucosa. J-chain-positive IgA cells that migrate to these areas may he stimulated by secondary’ signals, such as specific antigens. T cells, or hormones. This will lead to the production of secretory IgA
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lilliL .
Epithelium
•
•
Sub-epithelium
ú
•
J iliH . » •
IgG IgA IgM
(50) (50) (1)
igo
(1)
•
•
CD8 B cells CD4 (57) CD8 (22) B cells (21) CD4 (39) CD8 (50) B cells (11)
Glandular area IgM (2) IgD (2)
IgA (80) IgG (20)
@
Deep Vascular area
CD4 (57 CD8 (30 B cells (12
Fig. 7. I his diagram summarizes the distribution of immunocom petent cells (%) in the nasal mucosa [adapted from W intheret at.. 27. and Brandtzacg. 24], There is a preponderance of T helper cells in the epithelium: however, in the intcrglandular area, the CD8 cells, or cytotoxic suppressor cells, predominante. The distribution of immu noglobulins. according to Brandtzacg [24] is also summarized. Whereas in the subepilhelium the distribution for IgG and IgA is approximately equal, there is a marked predominance of IgA immu nocytes in the intcrglandular area.
in about 70% of IgG immunocytes and in almost 100% of IgD immunocytes [24], The Nasal Mucosa as an Immunocompetent Tissue Another immunological function of the nasal mucosa may be related to the production of local antibody to incoming inhaled antigen. As mentioned above, it is likely that most antigens are prevented from entering into the nasal mucosa by so-called ‘immune exclusion'. Specifically, such organisms as NTH I and Streptococcus pneumoniae are inhibited from adhering to both the nasopharyngeal and the nasal mucosas [25]. Specific IgA present in external may play an important role in the pathogenesis of otitis media in children. In general, all of the immunocompetent cells necessary for the induction of specific antibody are present in the nasal mucosa. The distribution and number of lympho cytes, monocytes, and cells expressing I1LA-DR antigen have been studied in a number of investigations in healthy adults using monoclonal antibody avidin-biotin immuno peroxidase techniques. A summary of the findings of Whi ther et al. [27] and Brandtzacg |24] is graphically summa rized in figure 7. In general, T helper cells appear to be more predominant in the superficial layers of the muco
Mucosa! Immunology
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and molecular matter, not only by the mechanical cleaning of the mucociliary system, but by the local immune mechanism of secretory IgA. In addition to secretory IgA. there arc other nonspecific mechanisms that the nasal mucosa has available to it. including the release of lactoferrin and lysozyme [24]. Lactoferrin is an iron-binding pro tein which exerts a bactericidal and bacteriostatic effect on both gram-positive and gram-negative bacteria, as well as Candida. Lactoferrin also appears to be enhanced by secre tory IgA. Lysozyme, or muramidase. is also bactericidal and bacteriostatic for certain gram-positive bacteria and accelerates the lytic effect of antibody-activated comple ment on certain gram-negative bacteria which may also involve secretory IgA. Both lactoferrin and lysozyme seem to be selectively produced by the serous glandular element and are absent from the mucus glandular elements of the nasal mucosas. In addition to secretory IgA. which is the major immu noglobulin that is active in immune exclusion, other immu noglobulin isotypes are present in the nasal mucosa. IgG is present primarily in the lamina propria, whereas secretory IgA is primarily present around the glandular elements deeper in the mucosa [24]. However, there is a great deal of IgG which is present in the nasal mucosas as a transudate from the serum. It has been demonstrated by a number of investigators that in inflammatory rhinitis, there is an increase in the number of IgG-producing immunocytes and a decrease in the number of IgA immunocytes [24], In nor mal nasal mucosa, Brandzaeg [24] has shown that 76.7% of IgA is gland-associated, whereas 46% is surface-associ ated. whereas in inflammatory rhinitis, approximately 62% of IgA is gland-associated and 24% is surface-associated. In addition to secretory IgA and IgG. the nasal mucosa pos sesses many more IgD immunocytes than any other secre tory mucosal surface in the body, whereas in the gastro intestinal tract. lgM appears to be more predominant. IgEproducing immunocytes are virtually absent from normal nasal mucosa but are apparently somewhat increased in allergic nasal mucosa. There is still a significant controversy as to whether most of the IgE present in the nasal mucosa is associated with mucosal and connective tissue mast cells rather than IgE-producing immunocytes. In summary, the glandular areas of the nasal mucosa represent an extremely potent site of IgA formation, whereas little IgM is produced locally. On the other hand, compared with normal intestinal mucosa and exocrine glands, a relatively large amount of IgG is produced in the mucosa even in the absence of inflammation. A remarkable number of IgA and IgM cells in the nasal mucosa arc J-chain-positive. J chain is present in free form
32
16 Antibody Titer 8 ya
•
•
••
•
tm
mi
— •—
— •—
—
•
•
• m
4 2
T Live Vaccine
18
1 22
26
• 30
1 34
Months
Fig. 8. Levels of SIgA poliovirus type I antibody in the nasopha rynx after primary immunization with trivalent live attenuated polio virus vaccine in healthy children whose tonsils have not been removed. The individual and mean antibody titers are shown. The antibody titers maintain their levels for a period of at least 34 months.
Fig. 9. Level of IgA poliovirus antibody in the nasopharynx before and after tonsillectomy and adenoidectomy (T&A). The chil dren were immunized with live polio vaccine 6 months to 6 years before the operation. Individual and mean geometric mean titers arc shown. The mean antibody titers decrease three-fold after the removal of tonsils and adenoids from 11.8 before and 3.9 after the operation (p
