| 1. |
- Bas, Anna, 1973-
(författare)
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Extrathymic T cell receptor gene rearrangement in human alimentary tract
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2+CD7+CD3-, CD4+CD8+, CD1a+, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2+CD7+CD3- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells.The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel.Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD.Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.
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| 2. |
- Fahlgren, Anna, 1972-
(författare)
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Defence capabilities of human intestinal epithelial cells
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins.To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry.Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms.Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells.The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression.Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding.We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation.Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin
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| 3. |
- Ou, Gangwei, 1958-
(författare)
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Human intestinal epithelial cells in innate immunity : interactions with normal microbiota and pathogenic bacteria
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rod-shaped bacteria were previously shown to be associated with the small intestinal epithelium of children with celiac disease (CD). Using culture-dependent and independent methods, we characterized the microbiota of small intestine in children with CD and controls. The normal microbiota constitutes an unique organ-specific biofilm. Dominant bacteria are Streptococcus, Neisseria, Veillonella, Gemella, Actinomyces, Rothia and Haemophilus. Altogether 162 Genus Level Operational Taxonomic Units (GELOTU) of six different phyla were identified in a total of 63 children. In biopsies collected during 2004- 2007 we did not find major differences in the microbiota between CD patients and controls. However, in biopsies collected earlier from children born during the “Swedish CD epidemic” and demonstrated to have rod-shaped bacteria by electron microscopy, we found that unclassified-Clostridales and Prevotella species were associated with CD. These anaerobic, rod-shaped bacteria showed marked affinity for the intestinal epithelium. Changes in breast-feeding practice and/or regiments for introduction of gluten containing food probably affect the composition of the bacterial flora in small intestine. We hypotesize that these bacteria contribute to contraction of CD.An in vitro model for studies of immune mechanisms of the intestinal epithelium was established. Polarized tight monolayers of the human colon carcinoma cell lines, T84 and Caco2, were developed by culture in a two-chamber system. The two cell lines showed the features of mature- and immature columnar epithelial cells respectively. Polarized monolayers were challenged with bacteria and proinflammatory cytokines. Immune responses were estimated as quantitative changes in mRNA expression levels of a secreted mucin (MUC2), glycocalyx components (CEACAMs, MUC3), antimicrobial factors and cytokines (IFN-g, TNF-a, IL-6 and IL-8). Tight monolayer cells were more resistant to bacterial attack than ordinary tissue culture cells and only B. megaterium induced the defensin, hBD2. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-a induced markedly increased levels of IL-8 and TNF- a itself in both cell lines suggesting recruitment and activation of immune cells. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-g was selective for CEACAM1- L while TNF-a upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.As a pathogenic enteric bacterium, Vibrio cholerae secretes cholera toxin that is the major factor of cholera diarrhea. However, some strains of O1 serogroup lacking the cholera toxin still cause enterocolitis and most V. cholerae vaccines candidates exhibit reactogenicity in clinical trails. An extracellular metalloprotease PrtV was characterized. It was associated with killing of bacteria predators such as the nematode Caenorhabditis elegans. Its role in human intestine was addressed by using the T84 tight monolayer in vitro model. We found that Vibrio Cholera Cytolysin (VCC), a pore-forming toxin, induces an inflammatory response in intestinal epithelial cells that includes increased epithelial permeability and induction of IL-8 and TNF-a and hence could be responsible for enterocolitis. The inflammatory response was abolished by PrtV thus VCC is indeed an autologous substrate for PrtV. In protein rich environment PrtV degradation of VCC was inhibited, suggesting that the magnitude of the inflammatory response is modulated by the milieu in the small intestine. Thus, VCC is likely to be part of the pathogenesis of cholera diarrhea and the causative agent of enteropathy in V. cholerae strains lacking the cholera toxin.
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| 4. |
- Lundqvist, Carina
(författare)
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Human intraepithelial lymphocytes : a comparative study of phenotype, morphology, and functional properties of intraepithelial lymphocytes in gut and oral mucosa
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human intraepithelial lymphocytes (IEL) constitute a unique cell population situated in the first line of defense of the alimentary tract. Here they are continuously exposed to a massive antigenic load of high complexity. However, different conditions prevail along the alimentary tract. In small intestine food antigens dominate whereas bacterial antigens are abundant in large intestine. The oral cavity is exposed to an enormous variety of antigens from the microflora as well as food constituents. The abundance and selective localization of lymphocytes in the surface epithelium of these challenged tissues implicate important roles for IEL in immune protection.IEL in normal human jejunum, ileum and colon as well as in normal and chronically inflamed gingiva were studied in situ and after isolation, with regard to phenotype, ultrastructure, cytokine mRNA expression and response to T-cell mitogens. Furthermore, an isolation technique was developed which yielded highly purified, functionally active IEL and enterocytes from the same sample.Intestinal IEL were situated in the basal part of the epithelium, often in small clusters and in close contact with adjacent lymphocytes and epithelial cells. They had an irregular shape with long processes and some had pseudopodium-like extensions penetrating the basement membrane. This indicates cell co-operation within the epithelium, as well as transmigration of IEL to underlying tissues. Freshly isolated IEL expressed several cytokines (IL-1β, IL-8, IL-2, TNF-α and IFN-γ) and in vitro activation induced expression of IL-2, IL-10, IFN-γ, TNF-α, TNF-β and TGF-β1, suggesting that IEL are involved in cell mediated cytotoxicity and suppressor cell activities.γδ T cells showed preferential homing to the epithelium both in gingiva and in intestine. They constituted the major lymphocyte population in normal gingiva and on average 30% of IEL at all levels of the intestine. Gingival as well as intestinal γδ IEL showed preferential usage of Vδ1Vγ8, suggesting common reactivity patterns along the alimentary tract. Intestinal γδ IEL and γδ IEL in normal gingiva were CD4-CD8-. In contrast, γδ IEL in chronically inflamed gingiva were predominantly CD8+ and showed induced expression of CD45RO. This indicates that γδ IEL participate in anti-bacterial immune responses in mucosa. Intestinal and gingival γδ IEL displayed ultrastructural features of cytolytic effector cells, e.g. electron-dense cytoplasmic granules and multivesicular bodies. They also expressed cytokines indicative of cell mediated-cytolytic effector functions. γδ IEL from inflamed gingiva expressed IFN-γ, TNF-α, TGF-β1 and IL-6 mRNA while intestinal γδ IEL expressed IL-2, IFN-γ and TNF-α.Intraepithelial αβ T cells were rare in gingiva while they constituted the major population of intestinal IEL. The phenotype of αβ IEL varied at different levels of the intestine. Thus, CD8+αβ IEL dominated in jejunum while cells with the unusual T-cell phenotype, CD4-CD8- TCR αβ+, constituted a major population of colonic IEL. CD4+ αβ IEL were equally represented, as a minor population, at all three levels of the gut. Intestinal αβ IEL had the same cytokine profile as γδ IEL. Taken together, these data suggest that αβ IEL are involved in immunoregulatory responses to luminal antigens.IEL with thymocyte-like phenotyped (CD2+TCR/CD3-, CD1+TCR/CD3-, CD1+TCRαβ+ and CD1+TCRγδ+) were present in jejunal epithelium. Furthermore, recombination activating gene-1 (RAG-1) mRNA was expressed in CD2+TCR/CD3- and CD3+/TCR- jejunal IEL. RAG-1 was not expressed in colonic IEL. Thus, the epithelium of small intestine is a site for extrathymic T cell maturation in humans.
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| 5. |
- Hultmark, Dan, 1949-
(författare)
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Insect immunity : Inducible antibacterial proteins from Hyalophora cecropia
- 1982
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A powerful bactericidal activity can be induced in the hemolymph of many insects as a response to an injection of bacteria. The nature of the effector molecules of this immune response was investigated, using pupae of the Cecropia moth, Hyalophora cecropia. Three major types of antibacterial proteins were found: the cecropins, the P5 proteins, and lysozyme. They appear in the hemolymph as a result of de novo synthesis.Six different cecropins were purified and characterized. The full amino acid sequences of the three major cecropins A, B and D were determined, as well as partial sequences of the three minor cecropins C, E and F. The cecropins are all very small (Mr = 4,000) and basic (pI > 9.5) proteins, and they show extensive homology in their sequences. The three major cecropins are products of different genes. Their C-terminals are blocked by uncharged groups, which can be removed by mild acid hydrolysis. The minor cecropins are closely related to the major forms, and may be unblocked precursors or, in one case (cecropin F), a minor allelic form. The cecropins were shown to be lytic, and to be efficient against several Gram positive and Gram negative bacterial strains, but not against mammalian cells.The P5 proteins are bactericidal proteins, larger than the cecropins (Mr = 20,000 - 23,000). Six forms, differing in isoelectric point, were isolated. They form two closely related groups, the basic (P5 A-D) and the acidic forms (P5 E-F). Within each group, the different forms have almost identical amino acid compositions.The Cecropia lysozyme is similar to lysozymes isolated from other insects, as well as to that from hen egg white. It is lytic to a restricted number of Gram positive bacteria.The presence of cecropins and other antibacterial factors was demon-strated also in other lepidopterans, notably Galleria mellonella, and may explain earlier observations of antibacterial factors in the latter species.
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| 6. |
- Mincheva-Nilsson, Lucia
(författare)
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Immune cells in pregnant uterine mucosa : functional properties, cellular composition and tissue organization
- 1993
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pregnant uterus mucosa - decidua - is an "immunologically privileged" site. A semiallogeneic embryo is allowed to survive, develop, and grow while the same tissue implanted outside the uterus will be rejected. The decidua basalis, which participates in the placenta formation, is a tissue rich in lymphoid cells. We have studied decidua associated mononuclear cells (DMC) from normal early pregnancies in humans. The cells were investigated with respect to surface marker profiles, ultrastructure, organization in the tissue, and functional properties. In addition, we have studied the expression of receptors for the iron-binding protein lactoferrin on these cells, and characterized the receptor (Lf-R).Ten to fiftee percent of all cells in decidua belong to the lymphoid cell lineage. They are present in aggregates [lymphoid cell clusters (LCC) mainly located in the vicinity of decidual/endometrial glands] and as individual cells, intra- or subepithelially along the glands (IEL) , and in the stroma. The LCC appear to be centres of immune reactivity. They occur at a frequency of 0.40.2/mm2 tissue and are composed of different population of activated T cells and NK cells in close contact with each other. Interestingly, B cells are not present in the LCC. DMC consist of four major lymphocyte subpopulation of similar sizes: TCRγδ+/CD56+cells, TCRγδ+/CD56-cells, TCRγδ-/CD56+cells and TCRαβ+/CD8+cells. TCRαβ+/CD4+ cells and monocytes are also present. Most DMC have long, thick processes, microvilli, and cytoplasmic granules. They are in intimate contact with surrounding lymphoid, epithelial and stromal cells. Signs of cellular movement and excretion of granules are also seen.About half of the T cells are TCRαβ cells. These cells lack CD4 and CD8. A large fraction of them are CD56+, a rare phenotype at other sites. Most of the TCRγδ+ cells express activation/memory markers (CD45RO, the Kp43 antigen, transferrin receptor, and MHC class II antigens), and many cells express the mucosa homing receptor HML-1. Morphologically these cells either display features characteristic for cytotoxic cells or contain unique nuclear inclusions.More than half of TCRαβ cells are CD8+, but CD4+ cells are also found. These cells also display activation markers.DMC use both transferrin and lactoferrin for their iron supply. The Lf-R on activated lymphocytes appears to be made of two peptides of 47 and 65 kD MW.Freshly isolated DMC respond poorly or not at all to activation through the TCR/CD3 complex, probably due to the low surface density of the complex. However, the TCR/CD3 complex can be up-regulated by stimulation with PMA/Ionomycin in vitro, suggesting that the lymphocytes are suppressed in vivo. Glandular epithelial cells produce immunosuppressive factor(s) that act on CD8+, TCRγδ+, and CD56+ cells. The proximity between the LCC and the glands indicates that this factor(s) may play a role in local immunosuppression. The identity of the factor(s) is presently unknown. The cytokine mRNA profile of DMC, as determined by RT-PCR, reveals IFN-γ, IL-8 and TGF-β1 mRNA in all samples, and IL-1β, IL-2, IL-10, TNF-α and GM-CSF mRNA in some samples. The cytokine profile is compatible with down-regulation of CTL activity.The demands on the immune system in pregnant uterus mucosa are unique. On one hand, a genetically incompatible fetus must be accepted, the development of the placenta must be allowed, and the uteral mucosal tissue must be remodelled. On the other hand, the invasiveness of the trophoblast must be controlled, and the fetomaternal unit must be protected against infections. Our studies indicate that this is achieved through a highly regulated process involving different types of activated lymphoid cells interacting with each other and with glandular epithelial cells.
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| 7. |
- Yeung, Moorix Mo-Wai
(författare)
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Specific and nonspecific immune mechanisms in human gut : a comparative study of normal and ulcerative colitis intestine
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The intestine, with its large mucosal surface area, digests and absorbs food nutrients and maintains a beneficial microbial flora in the colon. Local protective immune responses against intestinal pathogens ensure the survival of the individual. These immune reactions are both specific and non-specific in nature. The intestinal epithelium is single-layered and constantly renewed with differentiating epithelial cells moving from the crypt to the luminal surface. Intraepithelial lymphocytes (IEL) are interspersed between the epithelial cells. Ulcerative colitis (UC) is a life-threatening chronic inflammation affecting colon.In this study three molecules belonging to the carcinoembryonic antigen (CEA) family, namely CEA proper, nonspecific cross-reacting antigen 50/90 (NCA 50/90) and biliary glycoprotein (BGP), were found to be specifically localized to the apical surface of colonic epithelial cells. Full expression of these molecules occurs when the cells reach the upper 1/3 of the crypts and are maintained on the mature cells at the luminal surface. Ultrastructurally, CEA, NCA50/90 and BGP are localized to microvesicles and microfilaments of the fuzzy coat/glycocalyx as well as to the microvilli of the epithelial cells. Their unique localization and documented bacterial binding capacities suggest that they have a role in innate immunity.Functional analysis of IEL in normal jejunum, ileum and colon revealed that IEL are in vivo activated T-lymphocytes expressing mRNA for the cytokines IL-1β, IL-2, IL-8, IFNγ and TNFα and that jejunal IEL have T-cell receptor (TCR)/CD3 dependent cytolytic capacity. As many as 10% of IEL actively produce IFNγ. CD4+TCRαβ+IEL, CD8+TCRαβ+IEL and CD4-CD8-TCRαβ+IEL all had a TH1/cytotoxic cytokine profile. IEL could be further stimulated in vitro to express IL-10, TNFβ and TGFβ1, to proliferate and to secrete IFNγ. Thus, active protection and/or regulation of the epithelium via cell-mediated immune reactions are prominent in the gut.UC colon was characterized by a marked lymphocyte infiltration in the lamina propria, 10-50 times the normal level. Most lymphocytes were present in follicle-like cell aggregates containing both T- and B-cells. An unexpected finding was that γδT-cells constituted about 15% of the cells in the aggregates. Such cells are only found intraepithelially in normal gut. γδT-cells of both the intestinal- (TCR-Vδ1/Vγ8) and blood type (TCR-Vδ2/Vγ9) were seen. T-cells in UC colon were activated but nonproliferating and had a down-regulated TCR/CD3 complex. RT-PCR and quantitative immunohistochemistry for cytokine mRNA (n=11) and protein respectively, revealed that the T-cells in UC colon did not produce IL-2, in marked contrast to T-cells in normal colon and to ileal T-cells from UC patients. This was a selective defect since TNFα, IFNγ, IL-1β, IL-8 and TGFβ1 were similarly expressed in normal colon. No TH2 cytokines were seen. Lamina propria leukocytes in UC colon expressed IL-6, a cytokine not found in normal colon. The epithelial cells of UC colon were activated expressing MHC class II antigens, heat shock proteins and the co-stimulatory molecule B7.1/CD80.Our study demonstrates that UC is an immunological disease. The immunopathological picture seen in UC colon probably reflects an inappropriate down-regulation of local immune responses perhaps due to a selective loss of the key cytokine IL-2 in a situation of extreme antigenic stress.
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| 8. |
- Olofsson, Katarina
(författare)
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Immune cells in human pharyngeal and palatine tonsils and in the uvula : tissue distribution, cellular composition and functional properties
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The adenoid (pharyngeal tonsil), the palatine tonsils and the uvula are strategically located at the entrance of the upper airodigestive tract. By virtue of their location they are incessantly exposed to inhaled and ingested antigens. Severe nasal obstruction, otitis media with effusion, recurrent tinsillitis and obstructive airways during sleep are conditions often due to diseases in these organs.To advance our knowledge about the etiology and pathogenesis of these diseases, we have compared immune cell composition, cytokine expression and microbial colonisation in adenoids from children with hypertrophic obstructive adenoid (HOA) and chronically infected adenoid (CIA). Similarly, we compared immune cell composition in palatine tonsils from children with idiopathic tonsillar hypertrophy and recurrent tonsillitis. Finally, we wanted to characterise the human uvula from an immunological point of view, which had previously not been done. Its composition and distribution of immune cells, its cytokine profile, connective tissue elements and ultrastructure was studied.When comparing adenoids from children with HOA and CIA, the most striking finding was their similarity. A cytokine profile that was independent of diagnosis but seemed characteristic for the adenoid emerged. T cell expression of IL-5 and TGF-β1 but not IL-4 suggested an ongoing humoral response driven by a "mucosal TH2" cell. αβ T cells also expressed TNF-α, IFN-γ and IL-2, indicating a concomitant cell mediated response. Cell mediated immune responses often reflect viral infection. In line with this, adenovirus DNA was found in 80% of the adenoid samples. Furthermore, IL-6, IL-8 and TNF-α expressed in the non-T cell fraction suggested that the tissue macrophages were activated. TNF-α, IFN-γ and TGF-β1 were expressed by γδ T cells. The following differences between HOA and CIA were however, noted: i) most intraepithelial lymphocytes were CD8+ γδ T cells in HOA, while CD8+ αβ T cells dominated intraepithelially in CIA; ii) the number of follicles was twice as high in CIA as in HOA; iii) there were signifacantly more granulocytes in the interfollicular area in CIA than in HOA; iv)IL-6 mRNA expressing γδ T cells were only found in HOA and v) there was a tendency of higher TNF-α mRNA levels in non-T cells of CIA compared to HOA. The following scenarios emerge: in CIA there appears to be an inadequate first line of defence, with a low frequency of intraepithelial γδ T cells and a high frequency of cytotoxic CD8+ αβ T cells eliminating infected epithelial cells. Togehter, these two conditions cause a "leaky" epithelium, allowing infiltration of microbes into the underlying tissue and subsequent recruitment of granulocytes and follicle formation initiated by activated macrophages. In HOA, activated intraepithelial γδ T cells appear to be involved in antimicrobial defence reactions and surveillance of the epithelium.The difference in leukocyte profiles between tonsils from patients subjected to surgery due to idiopathic tonsillar hypertrophy or recurrent tonsillitis was limited to the surface epithelium. CD8+ γδ T cells utilising the unusual combination Vδ1/Vγ9 in their T cell receptor constituted the majority of intraepithelial lymphocytes in both groups. However, the frequency of these cells was significantly higher in recurrent tonsillitis. These results suggest that CD8+ Vδ1/Vγ9+ γδ T cells are characteristic of palatine tonsils and selectively expanded in recurrent tonsillitis. These γδ T cells may be involved in clearing infectious bacteria at the surface of the tonsil.Tissue macrophages, αβ T cells, γδ T cells, mast cells and B cells constituted, in declining order, the immune cell populations in the uvula. No fillicle-like structures were present. Most T cells had a CD8+ CD28-TCR-αβ+ phenotype, suggesting a down-regulatory function. Production of the down-regulatory cytokine TGF-β was also noted. This is consistent with the hypothesis that the uvula contributes to the development of mucosal tolerance. Furthermore, the uvula seems to be protected from pathogens penetrating the internal milieu by a subepithelial barrier of γδ T cells and macrophages. TNF-α secreting immune cells were found at this location. TNF-α and TGF-β may cause tissue fibrosis, TNF-α indirectly by stimulating mast cells to release histamine. Tissue fibrosis in conjunction with water binding to hyaluronan present in the connective tissue is the most likely explanation for the observed enlargement of the uvula in patients with sleeping disorders.
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