Università degli Studi di Napoli Federico II Facoltà di Medicina e Chirurgia - PDF

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Università degli Studi di Napoli Federico II Facoltà di Medicina e Chirurgia Tesi di Dottorato di Ricerca in Fisiopatologia Clinica e Medicina Sperimentale (Coordinatore: Prof. G. Marone) XXII Ciclo

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Università degli Studi di Napoli Federico II Facoltà di Medicina e Chirurgia Tesi di Dottorato di Ricerca in Fisiopatologia Clinica e Medicina Sperimentale (Coordinatore: Prof. G. Marone) XXII Ciclo Cytotoxic activity of invariant NKT cells in human allergic contact dermatitis Tutor Prof. Fabio Ayala Candidata Dott.ssa Anna Balato 1 Index Introduction.pag. 4 Regulatory role of NKT cells pag. 8 Ligands pag. 9 Functional subsets of NKT cells..pag. 13 Antigen presentation and activation of NKT cells by non-professional APCs pag. 21 NKT and cytotoxicity.....pag. 23 Table 1.pag. 26 Figure 1...pag. 27 Materials and methods...pag. 28 Human subjects.pag. 27 RNA extraction, cdna synthesis, and real-time PCR.pag. 28 Reagents and antibodies.pag. 29 Immunohistochemical procedures.pag. 30 Primary KC, HaCaT, THP-1 and K562 cell lines.pag. 30 NKT-cell assay..pag. 31 Cytotoxic assays...pag. 31 Statistical analyses...pag. 32 Table 2 pag. 33 Results...pag. 34 Cytotoxic molecules gene expression in ACD.pag. 34 Co-localization of NKT cells and cytotoxic molecules in ACD..pag. 35 2 Gene expression during antigen presentation pag. 36 NKT cell cyototoxic activity vs keratinocytes...pag. 37 Discussion.pag. 38 References pag. 42 3 Introduction Natural killer T cells (NKT cells) are a unique subset of T-lymphocytes that express a T-cell receptor (TCR) a/ß, with a restricted repertoire (Godfrey et al., 2004). This distinguishes them from NK cells, although NKT cells share some markers characteristic of NK cells such as CD161 (CD161c is known as NK1.1 in mice) or NKR-P1 (Godfrey et al., 2004). In contrast to conventional T- lymphocytes and other Tregs, the NKT cell TCR does not interact with peptide antigen presented by classical major histocompatibility complex (MHC)-encoded class I or II molecules, but instead it recognizes glycolipids presented by CD1d, a nonclassical antigen presenting molecule that associates with ß2 microglobulin (Godfrey et al., 2000 and Kronenberg et al., 2002). Polymorphic MHC molecules present peptide antigens that are specifically corecognized by aß TCRs expressed on T-cells. These TCRs are highly specific and restricted to recognizing the MHC molecules of the individual from which they were derived; this concept is known as MHC-restriction. In contrast, CD1 family members are monomorphic MHC class-i-like glycoproteins that present lipid-based antigens recognized by T-cells, even across species. The antigen-binding cleft of the CD1 family contains large hydrophobic pockets that are ideally suited to binding lipid antigens and, on the basis of sequence identity and chromosomal location, are divided into CD1a, CD1b, CD1c, CD1d and CD1e family members. Although humans express all family members, only the CD1d genes are present and expressed in mice and rats. CD1a, b and c are most homologous to each other and are expressed by thymocytes, dendritic cells and activated monocytes, (Dougan et al., 2007) while 4 B cells express only CD1c, among these 3 family members (Small et al., 1987). CD1d is divergent in sequence from CD1a, b and c (Balk et al., 1989) and its tissue distribution is more widespread, including cells outside the lymphoid and myeloid lineages (Canchis et al., 1993; Exley et al., 2000). In addition CD1d is expressed in epidermal keratinocytes (KCs) in the skin, and by several other peripheral epithelia or stromal cells in diverse organs such as the intestine, liver, kidney, pancreas, uterus, and conjunctiva (Sieling, 2000). Some bone marrowderived cells, such as B cells and monocyte-derived dendritic cells, also express CD1d (Sieling, 2000). Thus, the wide distribution of CD1d in widely divergent tissues by predominantly nonprofessional antigen presenting cells (APCs) suggests that it plays an important, yet poorly defined, role in both health and disease states (Fishelevich et al., 2006). CD1a, b and c molecules, which can present self and foreign (microbial) lipid antigens, are generally recognized by polyclonal T-cells expressing aß TCRs or by some?d T cells that recognize CD1c. The recognition of glycolipid antigens in association with CD1d means that NKT cells recognize a class of antigens ignored by conventional T-cells. Relatively few examples of these antigens are defined, although presentation of several mycobacterial cell wall antigens by the 3 other human CD1 molecules, CD1a, CD1b and CD1c, has been well characterized (Gumperz et al., 2001), as has presentation of several autologous antigens, including gangliosides and some phospholipids. The compound most efficient for activating the majority of NKT cells is a synthetic glycolipid (originally derived from a marine sponge) known as a-galactosylceramide (agalcer) (Hayakawa et al., 2004 and Hong et 5 al., 1999). This compound binds effectively to CD1d, and the complex of glycolipid plus CD1d binds the NKT cell TCR (Sidobre et al., 2002). agalcer is widely used as a highly specific antigen for both human and murine NKT cells. In both species, these cells use precisely rearranged homologous TCR variable (V)a and junctional (J)a segments. In mice, NKT cells most express an invariant TCR a chain rearrangement (Va14- Ja18) with a conserved CDR3 region, and they typically coexpress either Vß8.2, Vß2, or Vß7. Similar T-cells are present in other mammalian species, including humans. The homologous population of human invariant NKT cells express a Va24-Ja18 rearranged TCR a chain with a Vß11-containing ß chain (Dellabona et al., 1994 and Porcelli et al., 1993). The evolutionary conservation of these cells is striking, as mouse NKT cells recognize human CD1d plus glycolipid antigen and vice versa (Brossay et al., 1998). NKT cells may be double negative for CD4/CD8 or may be single positive for CD4 or CD8. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Va14 NKT cell lineage via interactions with CD1d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Va14 NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NK1.1 and members of the Ly-49 inhibitory receptor family (MacDonald HR et al., 2007). The distribution of NKT cells and NK cells is different; NKT cells are found everywhere that conventional T-cells are found. In mice, NKT cells represent 6 approximately 30% of the total lymphocytes in the liver (50%of aßtcr+ T cells), 20% of the aß T-cells in the bone marrow, and 3% of the aßt cells in the spleen, but are rare in the lymph node (Berzins et al., 2004; Emoto & Kaufmann, 2003; Godfrey et al., 2000). In humans, only 0.2% of peripheral blood T-cells are NKT cells. They are also present in the human liver but their numbers are lower than in the liver of mice (Prussin & Foster, 1997; Benlagha et al., 2000; Norris et al., 1999). NKT cells predominate in the liver, while conventional T cells prevail in blood and lymph node. In contrast, the order for NK cell frequency is lung liver peripheral blood mononuclear cells (PBMCs) spleen bone marrow lymph node thymus, where NK cells are almost undetectable (Gregoire et al., 2007). NK cells are lymphocytes that play a vital role in cell-mediated immunity, and serve as the first line of defense against cancerous and virally infected cells. Their cytolytic activity is not regulated by antigenic specificity, but through a balance of activating or inhibitory signa ls mediated through cell surface receptors. These receptors specifically bind human leukocyte antigen (HLA) ligands (Bashirova et al., 2006), which are expressed in virtually all nucleated cells but downregulated in aberrant cells, thus allowing NK cell reconnaissance. This mode of recognition was first referred to as the missing self hypothesis (Ljunggren et al., 1990). Although most NKT cells express NK cell markers such as CD161, they also contain a small population of cells that are negative for NK cell markers (Godfrey et al., 2004). Importantly, CD1d-restricted T cells also contain T-cells that neither express the canonical TCR a-chain nor respond to agalcer (Duarte et al., and Terabe et al., 2005). To avoid confusion, it has recently been recommended that NKT cells should be defined by their reactivity to agalcer loaded onto CD1d multimers, instead of expression of NK cell markers (Godfrey et al., 2004). It is apparent, now, that the simple classification of NKT cells as T-lymphocytes that also express NK receptors is inadequate. Robson MacDonald reviewed the discovery of NKT cells through time. Early in the history, there was no connection with the NK cell lineage for either Vß8- biased DN thymocytes or Va14-expressing peripheral T-cells. He reported that this could probably be traced to the fact that only T-cell biologists were studying these rare populations and there was no reason at the time to suspect any shared markers between T and NK cells. On the other hand, NK cell biologists often were required to exclude T-cell markers from their analysis, putting them in a better position to identify shared phenotypic properties (Robson MacDonald, 2007). Around 1990, it was recognized that a subset of lymphocytes in spleen and bone marrow shared NK and T-cell markers (Yankelevich et al., 1989; Sykes, 1990). Importantly, this was also shown to be true for the DN Vß8-biased thymocyte subset (Ballas & Rasmussen, 1990). At this point in time NKT cells finally had a birth certificate but still were not linked to expression of the Va14 chain. Regulatory role of NKT cells NKT cells influence and regulate a wide range of immune responses. They share some characteristics with CD25+CD4+ Tregs, which, also regulate different types 8 of immune responses and which also have recently attracted much attention (Godfrey and Kronenberg, 2004). CD4+CD25+ Tregs are essential for the control of immune responses in inflammatory, autoimmune, or cancer diseases, and it is well established that in particular the lineage-specific transcription factor Foxp3, as well as cytokines (including IL-2, IL-10, and TGF-ß), characteristic surface markers such as CTLA- 4, and members of the TNF superfamily (e.g., RANKL) are critically involved in the thymic development, peripheral maintenance, and suppressive activity of CD4+CD25+ Tregs (Kim, 2007). There are some clear similarities between NKT cells and CD4+CD25+ Tregs, including expression of CD25 by human CD4+ NKT cells (Lee et al., 2002). This raises the possibility of confusion between these distinct types, such that some activity ascribed to CD25+CD4+ Tregs might really be due to NKT cells. It is therefore important to carefully distinguish these cells (Godfrey and Kronenberg, 2004). In Table1 similarities and differences among NKT cells, NK cells, T cells and CD25+CD4+ Tregs are summarized. Ligands Since agalcer, was discovered as a potent ligand for NKT cells (Kawano et al., 1997), a synthetic agalcer has widely been used for study of NKT cells as a surrogate ligand. It is now established that two lipid chains of agalcer are inserted to hydrophobic grooves of the CD1d glycoprotein expressed by APCs (McCarthy et al., 2007), whereas the a-linked sugar moiety is accessible and 9 recognized by the TCR of NKT cells. Recently, the crystal structure of the invariant TCR and CD1d loaded with agalcer has shown a very unique orientation of TCR towards CD1d (Borg et al., 2007), which allows a selective involvement of the invariant a-chain for recognition of the a-linked sugar. Comparison of agalcer with its structurally altered analogues has provided important insights into how NKT cells may differentially respond to glycosphoingolipids with lipid tail variants (Brutkiewicz, 2006; Miyake & Yamamura, 2007). An agalcer analogue called OCH (Miyamoto et al., 2001; Oki et al.,2004, 2005), with a shorter sphingosine, would selectively stimulate IL-4 production from NKT cells, whereas agalcer stimulation induces both IL-4 and IFN-?. Accordingly, OCH stimulation of NKT cells favors a Th2 bias of immune responses in vivo, as compared to agalcer stimulation. a-linked sugars such as agalcer are not recognized as a product of mammalian cells, implying that agalcer is not a physiological ligand for NKT cells. Currently, it is well recognized that NKT cells can be activated during infectious diseases (Tupin et al., 2007). Interestingly, it has been reported that agalcer-like glycosphingolipids are rather ubiquitously found in the environment, indicating that agalcer may be actually derived from bacteria residing with the marine sponge. Other analogs of agalcer have been shown to have various effects on NKT cell activation in vitro, as well as antimicrobial and antitumor activity in vivo. For example, modifying the acyl chain of the agalcer molecule KRN7000 to create a diunsaturated 20 carbon chain results in IL-4 production with a reduction in IFN? secretion (Yu et al., 2005). In contrast to the effects of acyl chain modification on 10 agalcer activity, Gonzalez-Aseguinolaza and colleagues used a different approach. In their studies, they used a C-glycosidic form of agalcer in analyses of the adjuvant properties of agalcer. It was found that the C-glycoside (a-c- GalCer) induced more of a Th1 response, was longer lasting, and was actually a better adjuvant than the parental compound itself in murine models of malaria and metastatic melanoma (Gonzalez-Aseguinolaza et al., 2000 and 2002; Schmieg et al., 2003 and 2005; Yang et al., 2004). Ortaldo et al (Ortaldo et al., 2004), as well as Parekh et al (Parekh et al., 2004), have found that a 12 carbon acyl chain form of galactosylceramide (ßGalCer (C12)) can stimulate NKT cells in a CD1d-dependent manner. The administration of the ßGalCer (C12) in vivo caused the apparent loss of NKT cells as found with agalcer, but without cytokine expression or activation of NK cells. Tetramers loaded with ßGalCer (C12) could stain NKT cells, although lower numbers were detected as compared with when agalcer-loaded tetramers are used (Wilson et al., 2003; Crowe et al., 2003; Harada et al., 2004). However, some contamination with minute amounts of agalcer cannot be completely ruled out in either of the ßGalCer preparations. Thus, ßGalCer may have some similar yet distinctly different actions on NKT cells. In 2005 three groups independently found that a predominant glycosphingolipid from the LPS-free Sphingomonas (S. capsulata, S. paucimobilis, and S. wittichii) and Ehrlichia muris bacteria could stimulate human and/or murine NKT cells in a CD1d-dependent manner (Kinjo et al., 2005 and 2006; Mattner et al., 2005; Sriram et al., 2005; Wu et al., 2005). All of these groups identified 11 aglucuronosylceramide (GSL-1) as this glycosphingolipid, with agalacturonosylceramide (GS-1`) also able to activate NKT cells. An additional unique aspect of GSL-1 as opposed to agalcer is the NKT cell populations identified by CD1d1 tetramers or dimers loaded with this glycolipid. Approximately one-half of those NKT cells that are agalcer/cd1d1 tetramer positive are GSL-1/CD1d1 dimer (or tetramer) positive. Two other sources of microbial products have been shown to be able to activate NKT cells. The Schaible group reported that Phosphatidylinositol mannoside (PIM) could stimulate both human and murine NKT cells (Fischer et al., 2004). Additionally, PIM-loaded CD1d tetramers could stain both human (PBL) and mouse (liver) NKT cells, although the latter detected a substantially smaller population than CD1d tetramers loaded with agalcer. Despite this interesting observation clearly identifying PIM as a CD1d ligand, how PIM actually participates in the immune response against mycobacteria is not yet clear. Search for an endogenous ligand of NKT cells has led to the identification of lysosomal glycosphingolipid isoglobotrihexosylceramide (igb3), a ß-linked sugar capable of stimulating NKT cells as a potential endogenous ligand for mouse and human NKT cells (Mattner et al., 2005; Zhou et al.,2004). With regard to the role of igb3 in adaptive immune responses, Mattner et al. reported that Gramnegative, LPS positive Salmonella typhimurium activates NKTcells through the recognition of igb3, presented by LPS-activated dendritic cells. However, very recent works have cast doubt on the meaning of the igb3 discovery (Porubsky et al., 2007; Speak et al., 2007). The study by Zhou et al. 12 (2004) indicated that igb3 presented by CD1d-expressing CD4+CD8+ thymocytes should be involved in the thymic positive selection of NKT cells. Porubsky et al. has then generated igb3 synthetase deficient mice and examined if NKT cells are really missing in the mice lacking expression of igb3. They found that the number and function of NKT cells were as normal as those seen in wildtype mice. Using highly sensitive HPLC assay, Speak et al. sought for the presence of igb3 in various mouse and human tissues. The only tissue containing igb3 was the dorsal root ganglion of mice. No igb3 was detected in any human tissue (Porubsky et al., 2007; Speak et al., 2007). These new findings do not support the idea that igb3 is central in the selection of NKT cells and reopened the search for endogenous ligands for NKT cells. Two more natural lipid components of the cell membrane, the disialoganglioside (GD3) and Phosphatidylinositol (PI), have been shown to be CD1d presented Ags capable of stimulating (at least some) NKT cells (Brutkiewicz, 2006). Functional subsets of NKT cells During their development in the thymus, conventional aßt-cells acquire the expression of either CD4 or CD8, MHC coreceptors that define functionally distinct MHC-restricted T-cell subsets. In response to antigen stimulation, CD4+T-cells differentiate into T helper, Th1 or Th2 cells. Th1 cells produce the signature cytokine interferon IFN?, whereas Th2 cells produce interleukin IL-4, IL -5, IL-13, and IL -10. Similarly, effector CD8+T cells can be classified as Tc1 or Tc2 cells, which produce type 1 or type 2 cytokines, respectively (Woodland & 13 Dutton, 2003). The Th1/Th2 paradigm has recently been reevaluated to include a third population of T helper cells, producing IL -17 and designated Th17. These proinflammatory T cells have been mostly studied in the mouse. Th17 cells exhibit a cytokine profile distinct from Th1 and Th2 cells, producing cytokines, such as IL- 17 and IL -22, and express a characteristic transcription factor retinoic acid receptor (RAR)-related orphan receptor gamma t (ROR?t). The differentiation of Th17 cells requires the coordinate and specific action of the proinflammatory cytokine IL-6 and the immunosuppressive cytokine TGF-ß. In addition, the IL-12 family member IL-23 is involved in the maintenance of these cells (Korn et al., 2007). A pro-inflammatory role in autoimmune diseases has been reported for IL- 17; furthermore IL -17 has been shown to be important for host defense against pathogens such as Klebsiella pneumoniae and Bacteroides fragilis (Stockinger and Veldhoen, 2007). The detection of IL-17-producing T cells in humans with inflammatory diseases, such as multiple sclerosis (MS), contact dermatitis (CD), rheumatoid arthritis (RA), and Lyme arthritis (Steinman, 2007) suggests that in humans these cells have a proinflammatory role similar to that described in mouse models (Rautajoki et al., 2008). From this perspective, the cytokine profiles of T-lymphocytes are thought to reflect their functional activities. In Figure 1 a model of the differentiation of CD4+CD8+ T-cells and activation of NKT cells is depicted. NKT cells are different from functionally differentiated conventional a/ß TCR bearing T-cells in that they are autoreactive (i.e., they can recognize self- 14 glycolipids) and produce both Th1 and Th2 cytokines, including IL -4, IL-10, and IFN?, upon stimulation with their ligands (Taniguchi et al., 2003). Two major subsets of NKT cells with distinct effec
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