Apoptosis in the Homeostasis of the Immune System and in Human Immune Mediated Diseases

Current Pharmaceutical Design, 2008, 14, 253-268 253

1381-6128/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

Apoptosis in the Homeostasis of the Immune System and in Human Immune Mediated Diseases

A. Giovannetti#,1, M. Pierdominici#,2, A. Di Iorio3, R. Cianci4, G. Murdaca5, F. Puppo5, F. Pandolfi4,

R. Paganelli3,*

1Department of Clinical Medicine, Clinical Immunology and Allergy, University La Sapienza, Rome;

2Dept. of Cell Biology and

Neurosciences, Istituto Superiore di Sanità, Rome; 3Dept. of Medicine and Sciences of Aging, University G. d’Annunzio, Chieti, and

Ce.S.I., Foundation University G. d’Annunzio, Chieti; 4Department of Internal Medicine, Catholic University, Rome and

5Dept. of

Internal Medicine, University of Genoa, Italy

Abstract: The immune system has evolved sophisticate mechanisms controlling the development of responses to dangerous antigens while avoiding unnecessary attacks to innocuous, commensal or self antigens. The risk of autoimmunity is continuously checked and bal-

anced against the risk of succumbing to exogenous infectious agents. It is therefore of paramount importance to understand the molecular events linking the breakdown of tolerance and the development of immunodeficiency. Apoptotic mechanisms are used to regulate the de-

velopment of thymocytes, the shaping of T cell repertoire, its selection and the coordinate events leading to immune responses in the pe-riphery. Moreover, they are at the heart of the homeostatic controls restoring T cell numbers and establishing T cell memory. T lympho-

cytes shift continuously from survival to death signals to ensure immune responsiveness without incurring in autoimmune damage. In this review we shall consider some key facts on the relationship of lymphopenia to autoreactivity, the mechanisms controlling positive and

negative selection in the thymus, the role of apoptosis in selected primary immunodeficiency states and in systemic and organ-specific autoimmunity, with examples from human diseases and their animal models.

Key Words: Thymus, immunodeficiency, apoptosis, selection, autoimmunity, homeostasis, cytokines.

INTRODUCTION

Apoptosis plays an essential role in T cell development, the shaping of the immune repertoire, and in the ordinate initiation and final resolution of immune responses to exogenous dangerous sig-nals [1]. Thymocytes expressing nonfunctional or strongly autore-active T cell receptor (TCR) specificities are eliminated by apopto-sis during development [2], and tolerance is centrally induced through ectopic expression of self antigens allowed by the autoim-mune regulator (AIRE) gene [3]. Apoptosis also leads to the dele-tion of expanded effector T cells during immune responses as well as the preservation of T cell memory and the general homeostasis of immune cells [4]. The dysregulation of apoptosis in the immune system results in immunodeficiency and autoimmunity [5].

IMMUNE HOMEOSTASIS

The numbers of T lymphocytes both in secondary lymphoid organs and in the circulation are kept under strict control [6], so that the CD3+ cell number is maintained even in the face of a sudden drop of a particular subset – as shown by the example of primary HIV infection [7, 8]. Homeostatic proliferation is the essence of this mechanism, which is called into action when lymphopenia occurs [9]. Lymphopenia induces proliferation of naïve T cells without CD28 costimulation [10] or antigenic stimulation, and in this condi-tion they acquire memory T cell markers [11]: however, not all lymphoid cells expand in this scenario, opening the door for replen-ishment by self-reactive cells [5, 12]. Interestingly, many autoim-mune diseases present with lymphopenia, and this is also oberved in animal models of human diseases [5, 13]. The reciprocal observa-tion that several activating factors stimulate T cell division and result in the generation of long-lived memory cells which reduce the incidence of autoimmunity, including bacterial antigens (adju-vants), chronic viral infections and mycobacterial species, repre-sents a cornerstone of the hygiene hypothesis of the rapid surge of immune mediated diseases in Western societies [5, 14, 15].

*Address correspondence to this author at the Dept. of Medicine and Sci-

ences of Aging c/o Pal. Se. Bi.Via dei Vestini, 66013 Chieti scalo (CH) –

Italy; E-mail: [email protected] #These authors contributed equally in the preparation of the manuscript.

Cytokines

IL-21 has been postulated to contribute to the generation of perturbed T cell homeostasis in autoimmune models of diseases [5, 12]. Part of its effects seems to be mediated by antagonizing the prosurvival effect of IL-15 on memory CD8+ T cells [12], decreas-ing Bcl-2 and Bcl-XL expression [16] thus targeting them for rapid elimination by apoptosis. In this context a temporary depletion of T cells in susceptible hosts would trigger homeostatic repopulation by peripheral tissue T cells when thymopoiesis is impaired and regula-tory function is inadequate [17]. When considered on the opposite side, both lack of IL-15 and IL-2 deficiency would result in devel-opment of autoimmune characteristics [18], as it is indeed observed in KO mice [19]. Also in these models, repopulation in the absence of T regulatory function [20] would give an advantage to a skewed anti-self repertoire. It is unclear at present which signals could drive homeostatic proliferation in the absence of these cytokines [21] essential for T lymphocyte growth.

It is interesting to note that mild depletion of T lymphocytes with modified anti-CD3 monoclonal antibodies set the stage for new ways to manipulate the immune system in autoimmune type 1 (insulin-dependent) diabetes [22].

MOLECULAR SWITCHES FOR T CELL APOPTOSIS

During adaptive immune responses, as mentioned in the intro-duction, an expansion phase of T cells, differentiating into effector and memory subsets through asymmetric rounds of division [23], must be followed by a contraction phase to reach normal numbers [24]. This goal is reached by induction of apoptosis through activa-tion-induced and activated T cell autonomous cell death [25]. Sev-eral factors control the sensitivity to apoptosis induction during the different phases of the immune response [26], thus acting as mo-lecular switches between T cell survival and death. The engagement of the T cell receptor (TCR) distinguishes the two apoptotic path-ways, which both result in caspase activation. The molecular switches so far identified range from cytokines (mainly chain containing such as IL-2, IL-7, IL-15 and IL-21, but also IL-6 and IFN- ), death receptors (Fas and TNFR-I), Bcl-2 proteins, NF- B, other kinases and specific organelles (mitochondria and lysosomes). The understanding of the regulation and reciprocal interactions of

254 Current Pharmaceutical Design, 2008, Vol. 14, No. 3 Giovannetti et al.

these switches may lead to new ways to control activated T cells that have escaped apoptosis in autoimmune disorders [25].

In the following sections we shall delineate the intrathymic selection of the T cell repertoire with discrete steps enabling central mechanisms of self-tolerance [27], the aberrations of apoptotic mechanisms leading to autoimmune diseases in genetic defects such as autoimmune lymphoproliferative syndrome (ALPS) and other primary immunodeficiencies, and finally we shall offer some ex-amples of the role of apoptotic imbalance in intestinal immuno-mediated disorders such as inflammatory bowel disease (IBD) and celiac disease.

THE THYMUS IN T CELL DEVELOPMENT AND REPER-TOIRE SHAPING.

Thymus Colonization

In order to generate T cells, hemopoietic precursors are required to colonize the thymic microenvironment. Interestingly, the site of production of T cell precursors changes during ontogeny. Fetal liver-derived precursors colonize the early fetal thymus while the postnatal thymus is colonized by precursors coming from the bone marrow [28]. Differently from adult precursors, fetal precursors have less myeloerythroid potential and can differentiate in the ab-sence of IL-7 [29]. The reasons for these differences are not clear. A possible explanation is that fetal and adult precursors receive distinct prethymic maturational signals capable to regulate their lineage potential. In this regard it should be noted that the fetal liver microenvironment contains discrete Notch ligand-bearing stromal cells that could activate Notch signalling (a pathway strongly impli-cated in T/B lineage choice) in fetal precursors prior to thymus colonization [30] (Fig. (1)). Although the mechanisms allowing precursors to exit blood vessels and entry to the thymus are still undefined, several molecules have been implicated in this process including the CD34, MECA79, VCAM-1, ICAM-1, and P-selectin [31]. However, recruitment of precursors to the thymus is also a temporally regulated process, in which a key role might be exerted by changes in expression of adhesion molecules as well as by intra-thymic competition for stromal cell niches [32].

Interleukin-7

The essential role of IL-7 in T cell development is well estab-lished. Its specific receptor consists of two chains, IL-7R and gamma common ( c). The latter is also part of the receptors for fundamental cytokines such as IL-2, IL-4, IL-9, IL-15, and IL-21. Genetic defects involving the genes encoding for c [33], IL-7R [34], or the Janus kinase 3 (Jak3, a component of the IL-7-induced signal transduction pathway) [35] account for the majority of severe combined immune deficiencies (SCID) characterized by strongly reduced numbers or even absence of T cells (see following section). It has been reported that thymocytes from c-deficient infants pos-sess TCR D-J but lack V-DJ rearrangements [36]. This finding may explain the complete lack of T cells in many c- and IL-7R -deficient patients. Since IL-7 is also required for peripheral T cell homeostasis [37], it can be speculated that the absolute T cell defi-ciency in c-deficient patients is caused by a combination of defects in early T cell development and in T cell homeostasis. IL-7, is es-sential for survival through the induction of the anti-apoptotic MCL-1. IL-7R - or common chain -deficient animals exhibit a block of T cell development at the DN 2-3 stages, [38] Similarly, the T-cell-specific MCL-1 deficiency, results in thymic atrophy and enlargement of the DN compartment [39].

T Cell Progenitors (TPs)

TPs enter the thymus at the cortical-medullary junction. A pro-portion of the precursors that seed the human thymus are unques-tionably multipotential whereas is still unclear whether some of the precursors that migrate into the thymus are already lineage re-stricted before their entrance. Likely, CD7 and CD10 define the

CD34+CD45RA

+ precursors biased to lymphoid lineages. An analy-

sis of the TCR rearrangement status indicates that the most imma-ture population in the thymus is CD34

+ CD10+CD38

lo [40], proba-

bly the direct progeny of the CD34+CD45RA

+CD7

+CD10

+CD38

lo

cord blood cells that have T, B, NK and some GM precursor activi-ties. The presence of multipotential precursors in the human thymus indicates that T cell commitment takes place within this organ. Complete TCR and partial TCR (D -J ) rearrangements have been reported in CD34

+CD7

+ cord blood precursors [41] and T

cell–restricted precursors were identified in human bone marrow [42] but whether these cells can migrate to the thymus is still un-clear. ETP have a CD34

+CD1a

- phenotype. Downstream of the ETP

is the CD4 immature single-positive (CD4 ISP) [43] population which can be divided into CD4

+CD1a

+ (CD4 ISP

lo) and CD4

hi

CD1ahi

(CD4 ISPhi

) populations (Fig. (1)). The subsequent subsets are the CD4

+CD8

+ - (early double-positive) and CD4

+CD8

+ +

populations, the latter cells being the precursors of double-positive TCR

+ cells.

Thymic Microenvironment

The pool of self-epitopes available for repertoire selection com-prises intrathymically expressed ubiquitous antigens and antigens specific to various types of thymic APCs such as cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs), thymic dendritic cells, macrophages, and thymic B cells. Each of these cells presenting unique sets of self-peptides, contribute to the diver-sity of self-antigen displayed in the thymus. Self-antigens have access to the thymus also via the circulation or by association with immigrating cells [44]. However these latter routes are unlikely to allow for a comprehensive array of extrathymic tissue antigens. Therefore, mechanisms of peripheral tolerance, i.e. when the first encounter of a T cell with its cognate TSA occurs outside of the thymus, have been suggested in order to explain the absence of auto-reactivity toward tissue-restricted self-constituents.

Recent findings, showing that TSAs are expressed in the thy-mus and participate in selection of the T cell repertoire, have lead to a reconsideration of the capacity of central tolerance. It is now commonly accepted that expression of TSAs, in evident contraven-tion of the rules established for cell type–specific regulation of gene expression, is a natural property of TECs, and particularly of mTECs, a phenomenon known as promiscuous gene expression [45]. By inclusion of self-antigens that represent most if not all parenchymal organs [46], promiscuous gene expression widens the extent of central tolerance in a manner that was not expected by original concepts of self–non-self discrimination. However, with the notable exception of the involvement of the autoimmune regula-tor Aire, both molecular and cellular regulation of this unconven-tional gene expression pattern are still poorly understood.

Development of T Cells ( / -Lineage Choice)

Two major types of mature T cells are generated in the thymus that can be distinguished by the clonotypic subunits contained within their TCR complexes: T cells and T cells. T cells and precursors to the T cell lineage (bearing the pre-TCR) are thought to be derived from a common CD4

-CD8

- DN precursor

[47].

DN thymocytes that have successfully rearranged TCR- and TCR- chains and expressed a mature TCR are capable of differ-entiating along the -lineage pathway remaining in the DN stage [48]. As opposite, cells harboring in frame TCR- rearrangement and expressing a pre-TCR are capable to differentiate along the -lineage pathway, passing from the DN to the CD4

+CD8

+ DP stage.

Most T cells are DN, except for some intestinal epithelial lym-phocytes, which can express CD8 homodimers. Instead, -lineage cells express, with few exceptions, either CD4 or CD8 heterodimers [49].

Apoptosis in the Homeostasis of the Immune System and in Human Current Pharmaceutical Design, 2008, Vol. 14, No. 3 255

An unsolved matter is whether pre-TCR- and TCR-mediated signals dictate versus T cell lineage choice, and if so, how these signals differ. Two models have been initially proposed: the stochastic/selective and instructive models. According to the first the / -lineage commitment is determined independently of TCR signalling whereas the instructive model postulates that commit-ment is directly determined by the TCR isotype [50]. Now evidence exists supporting a model of lineage commitment based on the sig-nal strength whereby stronger signals promote development while weaker signals promote the fate, irrespective of the TCR isotype [51]. During early stages in T cell development, the TCR loci undergo rearrangement in a sequence TCR > > > [52] but it is still unclear in which subset the rearrangement of particular loci occurs. The current opinion is that expression of a TCR pro-tein and the resulting -selection occur within a certain develop-mental window and are not closely coupled to regulation of CD4, CD8 , and CD8 expression. Currently available data indicate the existence of a first limited number of cells that undergo the -selection before CD4 is expressed [52], a second, larger proportion of cells that is -selected after upregulation of CD4 [53], and finally a third group of the pre-T cells that upregulates CD4 and CD8 before initiating and completing TCR rearrangements [54] (Fig. (1)).

The Pre-TCR

The pre-TCR is initially expressed at the CD25+44

- DN3 stage,

following productive rearrangement of the TCR- locus. TCR- pairs with the pre-TCR- and molecules of the CD3 family forming a receptor that is capable to be expressed on the cell surface.

Pre-TCR signalling has the ability to regulate several transcrip-tion factors NF- B, NFAT [55], E2A/Id proteins [56], and -catenin/TCF-1 [57]. The expression of the pre-TCR receptor on the cell surface associates with the diminution of the IL-7R, Notch, and the hedgehog pathways, indicating that the pre-TCR may substitute the biological role of these pathways during T cell development. The pre-TCR differs from 'mature' and TCR in terms of both signalling initiation and biological outcome. The pre-TCR is a ‘weak’ signal transducer and that the commitment of immature DN cells to the lineage seems depends on these weak signals. Since all three receptors use identical signalling pathways and activate similar transcription factors there must be something unique about the pre-TCR that is not shared with the two other mature TCRs. As indicated by studies using transgenic mice expressing tailless pre-TCR forms, the main functions of the pre-TCR receptor are the pre-T cell proliferation and the development of a normally sized thymus [58]. In Pre-TCR-deficient mice thymi are small and the T cell development arrested at the DN3 stage. Pre-TCR-selected cells display a number of characteristics of cell-cycle entry including hyperphosphorylation of pRb, increased expression of cyclins A and B, downregulation of p27, increased cell CDK2 activity, and DNA synthesis. Unfortunately mechanisms of survival and apopto-sis in immature thymocytes, before the expression of antigen recep-tors and MHC recognition, are poorly understood. Probably, early thymic immigrants depend on prosurvival signals deriving from cytokine receptors for the upregulation of the anti-apoptotic Bcl-2 gene expression [59]. In the absence of these survival signals, the pro-apoptotic Bax translocates to mitochondria where it begins a death cascade.

Fig. (1). Developmental stages of intrathymic differentiation. The strong influence of Notch signalling in driving early precursors through DN stages 1-3a and

affecting the choice between or rearrangement is highlighted. After selection is made, only rearranged TCRB+ progress to DP through the immature

single positive (CD4 ISP) stage. Positive/negative selection occurs at the DP stage. Differential expression of several membrane antigens is shown to distin-

guish DN discrete maturative steps. TCR rearrangements are indicated in the lower part. Most extrinsic signals controlling the sequential entry are discussed in

the text, at each checkpoint failure to express more mature phenotype results in apoptotic elimination of thymocytes.


Signalling pathways inducing pre-T cell death in the absence of a pre-TCR are largely unknown. The initially proposed requirement of Fas-induced death pathway for pre-T cell death, has been re-cently challenged. Fas belongs to a subgroup of the TNFR family that interacts through their death domains with cytoplasmic adapt-ers like FADD and TNF-R1-ADD [60]. FADD functions as an adapter molecule in apoptosis initiated by Fas, TNFR-I, DR3, and TNF-related TRAIL receptors. On one hand, the demonstration that Rag

-/- FADD

-/- immature T cells can differentiate and reach the DP

stage indicates that FADD could be essential for the transduction of death signals resulting in pre-T cell death. On the other hand, FADD deficiency leads to inhibition of T cell development at the DN stage (with a consequent reduction in the number of mature T cells) and FADD mutation does not affect apoptosis or signalling events triggered by the pre-TCR [61]. Since the introduction of a TCR transgene failed to rescue the mutant phenotype, these obser-vations suggested that FADD is required for the proliferative phase of early T cell development.

The p53 pathway is involved in both cell survival and prolifera-tion. In fact, p53 regulate not only pro-apoptotic genes such as Bax, Bid, caspase-6, Puma, and Noxa but also cell-cycle regulators like p21

cip. It has been reported that DNA breaks resulting from recom-

bination events trigger death in pre-T cells through action of p53 whereas inactivation of p53 promotes the survival and differentia-tion of pre-TCR-deficient thymocytes [62], Therefore it can be speculated that pre-TCR regulates progression through the DNA-damage checkpoint of the DN to DP transition by inactivating p53.

Bcl-2 is expressed in the thymus, and it has been suggested to induce survival of immature lymphocyte progenitors. The central role of the mammalian pro-survival Bcl-2 proteins, like their virally encoded orthologs, is to inhibit Bax and Bak. Thus, BH3-only pro-teins trigger apoptosis indirectly through Bax or Bak by neutraliz-ing all of the relevant prosurvival proteins, allowing the activation of Bax and Bak to proceed.

However, it appears not to be essential at the stage of pre-TCR selection. Pre-TCR signalling induces Ca

+2 mobilization and activa-

tion of PLC kinase and NF- B transcription factor. Through the induction of genes whose products can inhibit cell death NF- B acts as an important regulator of pre-T cell survival. These genes include cellular inhibitors of apoptosis, caspase-8–c-FLIP and two members of the Bcl-2 family, Bcl-xL and BCL2A.

BCL2A1 (A1) is expressed at the DN thymic compartment and appears as an ideal candidate for the regulation of pre-T cell apop-tosis. Similarly to Bcl-2 and Bcl-xL, A1 contains all major homol-ogy domains of the Bcl-2 family [63], is developmentally regulated in the immune system and is induced on cellular activation [64]. When transfected into cell lines A1 slows cell death and heterodi-merizes with the pro-apoptotic Bax [65]. Moreover, A1 sequesters truncated Bid in order to inhibit its collaboration with pro-apoptotic Bak or Bax [66]. Overexpression studies showed that A1 could protect thymocytes from several death stimuli, including pre-TCR deficiency, suggesting an important role for this molecule in pre-T- cell survival [67].

Positive Selection and Negative Selection

A key step during T cell development is when immature CD4

+CD8

+ thymocytes undergo positive and negative selection

events based on the specificities of the TCR complexes they express. Thymocytes which recognize self-peptide/MHC com-plexes with a low avidity undergo a positive selection process re-sulting in rescue from cell death and the induction of a differentia-tion program leading to the generation of MHC class II-restricted helper CD4

+ T cells and MHC class I-restricted cytotoxic CD8

+ T

cells [68] . As opposite, thymocytes bearing potentially autoreactive TCR specificities that recognize peptide/MHC complexes with high avidity, undergo negative selection by the induction of apoptosis [69]. As a consequence of these combined processes, a self-tolerant

T cell pool capable of recognizing foreign antigens in the context of self-MHC molecules is generated.

Every day of postnatal life approximately 95% of the cells that reach the DP stage fail to become functional lymphocytes and die strengthening the concept that positive selection is a wasteful proc-ess. This highlights the critical role that positive selection plays in the establishment of a working immune system. With the positive selection is achieved the fundamental task for the development of T cells to match the large number of TCR specificities generated by gene rearrangement and the independent expression of a subset of MHC molecules from the polymorphic pool of MHC genes.

However, TCR recognition of self-MHC has been demonstrated in additional mechanisms including the regulation of mature naïve T cell survival [69], the homeostatic T cell proliferation [70] and the establishing of the threshold of T cell activation [45]. Therefore one can speculate that the stimulation of mature T cells by self-MHC may serve to maintain the two hallmarks of T cell recognition i.e. the high sensitivity for activation and the highly discriminate recognition of antigen. Positive selection is also a lineage diversifi-cation process, where randomly generated TCR specificities ex-pressed by DP cells are shunted into appropriate functional cell types. Such a process necessitates a TCR-mediated recognition event in order to distinguish expression of class I and class II MHC-specific antigen receptors that differ only in membrane-distal TCR-variable regions.

Negative selection often operates after positive selection and thus on the smaller population of thymocytes that have migrated to the cortico-medullary junction as part of their maturation process. Although the details of negative selection are scarcely understood it seems a process independent from positive selection and the associ-ated lineage commitment.

The first checkpoint, so-called beta-selection, is contingent upon pre-TCR signalling and ensures that only those DN thymo-cytes that have successfully rearranged their TCR locus progress to the CD4

+CD8

+ (DP) compartment.

For many years the removal of strongly self-reactive T cells has been viewed as the only purpose of negative selection. However, recent evidence drives now to reconsider in part this opinion indi-cating for negative selection a more basic role in molding the T cell repertoire. Huseby and colleagues [71], by limiting negative selec-tion, have demonstrated that after TCR rearrangement, most T cells display a very high affinity for MHC. Therefore, in addition to re-move undesirable clones, with the potential to drive autoimmunity, negative selection exerts an essential role in removing the T cells bearing TCR with the default, unmodified, high affinity to MHC. The survived T cells will be able to bind low affinity MHC, thus allowing the specificity of the adaptive immunity.

In spite of intensive investigations, the molecular mechanisms of clonal deletion remain to be fully elucidated [72]. This may be due to the different models developed for testing negative selection often involving different mechanisms.

The most broadly used models for negative selection are those of TCR transgenic mice expressing a receptor specific for a self-antigen along with that self-antigen.

These kinds of models may be divided according to the stage in which clonal deletion occurs. Among the possible explanations for why some transgenics delete early (at the DN stage) and others delete late (at the SP stage) there are:

1) the anatomy of self-antigen expression (DN cells reside in the subcapsular area or outer cortex and SP cells reside in the me-dulla);

2) the type of APC that presents the particular self-antigen;

3) the affinity of the TCR for self-antigen (DP thymocytes, ex-pressing TCR at lower level than SP might require a stronger TCR stimulus to die [73].


The injection of specific peptide into TCR transgenic animals also results in apoptosis of DP thymocytes [74] eliminating the concern about the artificial nature of cross-linking antibodies as TCR ligands. However, the massive activation of the cells respond-ing to the stimulus might cause nonspecific death of thymocytes [75] as well as other nonspecific effects such as a cytokine-mediated stromal cell–activation process [76], and the collapse of the thymic architecture [77].

Up to date the more physiologic models to study the antigen-induced clonal deletion are those involving TCR transgenic strain [78]. In fact this model does not present the nonspecific activation observed in other models. On the other hand, the reduced frequency makes the responders difficult to detect and study.

In some models clonal deletion does not occur when TCR transgenics are crossed to animals expressing the high-affinity ligand [79] . In these cases thymocytes expressing endogenously rearranged T cell receptors are preferentially generated. Two expla-nations have been proposed for such unusual findings: 1) the pref-erential survival of cells that had undergone secondary rearrange-ment prior to antigen encounter [80] and, 2) the upregulation of gene rearrangement signaled via the TCR [79]. The second hy-pothesis corresponds to the receptor editing process that occurs in developing B cells [81]. The reasons why deletion predominates in some models but not others are still undefined but could involve factors such as the timing and level of TCR expressed in different transgenics. Therefore, a key question is whether antigen-specific receptor editing occurs also in normal animals and whether some of the 30% of peripheral T cells that express two functional TCR rearrangements could be autoreactive.

Generation of T Regulatory Cells in the Thymus

The tolerance to self-antigens is realized in the thymus by mul-tiple mechanisms, including the generation of Treg and clonal dele-tion by apoptosis. Evidence exists indicating bone marrow-derived DCs and thymic epithelial cells as important mediators of thymo-cyte negative selection and Treg development, respectively [82, 83]. Dendritic cells expressing MHC class I and class II molecules are strategically located in thymus at the level of cortico-medullary junction. Here, they are supposed to screen the TCR specificities of positively selected CD4

+CD8

+ thymocytes during their migration

from the cortex to the medulla. However, negative selection may also occur when corticomedullary migration of thymocytes is ab-normal. It has been reported that the recruitment of new T cell pre-cursors to the thymus is accompanied by the migration of DCs, in order to ensure efficient negative selection during each wave of T cellprecursor development. Medullary epithelial cells might play a role in the recruitment and positioning of DCs in the thymus. In fact, mice deficient in Rel-b lack thymic DC cells, which has been shown to be a direct consequence of an absence of an organized thymic medulla [84].

The majority of Treg cells represent a separate, thymus-derived lineage of CD4 T cells [85]. Although different studies using TCR transgenic systems have demonstrated that the thymic epithelium plays an essential role for Treg development [83] in vitro studies [86] support a role for human thymic DCs in inducing Tregs. Data obtained in transgenic mice that likely express MHC class II mole-cules only on cTECs [86] suggest a cortical origin of the Treg line-age. In these mice both the number of CD4 SP thymocytes express-ing the CD25 marker and the suppressive function of peripheral CD4 CD25

+ T cells were similar to that observed in controls [87].

Therefore according to these data the self-antigen encounter in the medulla would be dispensable. However, the lack of rigorous con-trols (e.g. the capacity of isolated mTECs from K14-IA

b mice to

present MHC class II–restricted antigens in vitro) induce us to take with extreme caution these data. The timing and location of Treg commitment within the thymus has recently been analyzed using a mouse that expresses a fusion protein of FoxP3 and green fluores-

cent protein. FoxP3-reporter expression could be detected in thy-mocytes within the medulla but not in the cortex [88]. In support of a role of mTECs in the selection of Tregs are also a number of tar-geted mutations that, altering the medullary architecture and mTEC differentiation, lead to deeply reduced numbers of CD25

+ Tregs

[89]. However, extrathymic antigen encounter can induce the de novo generation of Treg from naive CD4+ T cells [90] qualitatively indistinguishable from that induced by mTECs in the thymus.

PRIMARY IMMUNODEFICIENCIENCY DISEASES AND

APOPTOSIS

Apoptosis, a physiological process of cell suicide, plays a piv-otal role in the maintenance of homeostasis of the immune system eliminating unwanted cells [91]. If decreased apoptosis can cause an overabundance of lymphocytes and possibly cancer as well as autoimmune and allergic reactions, enhanced apoptosis can result in lymphocyte depletion and immunodeficiency. Here, we will focus on the role of apoptosis in the pathogenesis of innate defects of the immune system, i.e. primary immunodeficiency diseases, and dis-cuss the importance of apoptosis modulation in both the pathogene-sis and treatment of these diseases.

Primary immunodeficiency diseases consist of a group of more than 100 inherited conditions which affect one or more components of the immune system [92-94]. Diseases in which lymphocyte func-tion is predominantly affected are commonly divided into three main groups, i.e. antibody deficiencies, cellular deficiencies and combined immunodeficiencies. In the antibody deficiencies, the genetic lesions mainly affects antibody production whereas in the cellular deficiencies cellular effector mechanisms are primarily compromised. In the combined immunodeficiencies both effector arms of specific immunity are impaired. This category contains the subgroup of SCID in which lymphocyte-dependent specific immu-nity is completely absent. Two other classes of primary immunode-ficiencies are also described: defects in phagocyte function and complement deficiencies. The clinical presentation of patients with a primary immunodeficiency reflects the type of defect present in the immune system and depends on the underlying genetic defect. Patients with SCID generally present with opportunistic infections and fail to thrive within a few months of life. Recurrent pyogenic infections are the hallmark of disease in patients with defects in B cells, phagocytic cells or complement, whereas opportunistic infec-tions with viruses or fungi are frequent in patients with T cell defi-ciencies. Primary immunodeficiencies frequently associate with autoimmune diseases, allergy and malignancies [95].

In the last 15 years the genetic basis of most primary immu-nodeficiencies has been identified. Many of these are associated with single gene defects, whereas others may be polygenic or repre-sent interactions of genetically determined characteristics with envi-ronmental factors [96, 97].

In some primary immunodeficiencies abnormalities of apopto-sis reflect a defect of gene(s) involved in the known mechanisms of cell death, e.g. Bcl-2 and Fas. In this category either diseases in which apoptosis is insufficient (e.g. autoimmune lymphoprolifera-tive syndrome) or enhanced (e.g. SCID) are included. In other pri-mary immunodeficiencies, such as Common Variable Immunodefi-ciency and Chromosome 22q11.2 deletion (del22q11.2) syndrome (DiGeorge syndrome/velocardiofacial syndrome) abnormalities of lymphocytes are likely to be an epiphenomenon of the ongoing immune activation operating in these disease.

Autoimmune Lymphoproliferative Syndrome (ALPS)

ALPS is one of the first human inherited disorders of apoptosis to be described and represents a failure of apoptotic mechanisms that help maintain normal lymphocyte homeostasis. It is character-ized by chronic, nonmalignant lymphadenopathy and splenomegaly, autoimmune manifestations, hypergammaglobulinemia, and expansion of circulating T cell receptor (TCR) /

+ CD4

-CD8

- T


cells [98-100]. ALPS generally associates with mutations of the Fas encoding gene (ALPS Ia) and rarely with mutations of FasL (ALPS Ib) [101-105], caspase-8 [106] or caspase-10 [107] gene (ALPS II). A number of ALPS cases lacking in any detectable molecular de-fects have been also described (ALPS III) [108]. In mice, the lpr and gld alleles cause similar diseases due to homozygous recessive mutations in the Fas and FasL gene, respectively [109-111]. Defec-tive apoptosis is the hallmark of peripheral blood lymphocytes of patients with ALPS [101]. Once activated, cultured T cells from ALPS patients exhibit reduced rates of apoptosis when stimulated through the T cell receptor CD3 or through the Fas pathway. ALPS patient B-cell lines transformed with Epstein-Barr virus show the same defective apoptosis after anti-Fas exposure, indicating that both B and T lymphocytes express the defect in Fas-mediated pro-grammed cell death [112]. In addition, mutation bearing family members, even those with no clinical abnormalities and no eleva-tions of CD4

-/CD8

- T cells, demonstrate defective apoptosis in vi-

tro. Thus, the impairment in cellular apoptosis associated with Fas mutation is inherited as a dominant trait.

The Fas/FasL pathway has a key role in the control of periph-eral tolerance and this accounts for the presence of autoimmunity in most ALPS patients as in Fas-deficient mice. Chronically stimu-lated T cells down-modulate CD28, a feature observed also in T lymphocytes of aged subjects [113], and therefore become more vulnerable to FasL-induced cell death according to either a suicide or a killing mechanism [114]. Fas-deficient T cells escape cell death, proliferate and can activate Fas-deficient B cells. In physio-logical conditions, FasL-expressing T cells kill autoimmune B-cell clones, likely because chronic exposure to antigens no longer in-duce protecting signals from cell death. Fas-deficient B cells will thus escape this regulatory process and further proliferate. CD40L/ CD40 T/B interaction can induce class-switch and further affinity maturation of immunoglobulins with autoimmune specificity [115].

Development of lymphoma has also been reported in patients with ALPS and their relatives [116-118]. Deregulated expression of Fas pathway may contribute to tumorigenesis and tumor escape from endogenous growth control. Fresh tumor cells and tumor cell lines, in fact, are often resistant to Fas-induced apoptosis, because they down-regulate Fas expression, secrete soluble forms of Fas, or express a Fas molecule with decreased signalling activity [119-123].

Indications for treatment of ALPS patients depend on the type and severity of symptoms [124]. Hypersplenism, anaemia, and/or thrombocytopenia may require therapeutic intervention. Glucocor-ticoids, intravenous immunoglobulin or cytotoxic drugs have been used with no or only transient effect. Van der Werff ten Bosch et al [125] reported reversion of ALPS with pyrimethamine (2;4-diamino-5-p-chlorophenyl-6-ethyl-pyrimidine), a folic acid antago-nist, suggesting a pro-apoptotic activity of this molecule. More recently, our group demonstrated that pyrimethamine, used at phar- macologically relevant concentration, induced per se apoptosis of activated lymphocytes via the activation of the caspase-8- and caspase-10-dependent cascade and subsequent mitochondrial depolari-zation, independently from CD95/Fas engagement [126] (Fig. (2)).

An ALPS-like clinical pattern (named autoimmune lymphopro-liferative disease [ALD]) has also been described by Dianzani et al [127] in patients with decreased Fas function, but no Fas gene mu-tation and peripheral expansion of CD4-

/CD8- T cells. ALD patients

display decreased apoptotic response to ceramide, triggering a death pathway partially overlapping the one used by Fas, which suggests that ALD is caused by downstream alterations of the Fas signalling pathway. Also this defect predisposes to the development of auto-immune diseases and cancer [128].

SCID

Mutations that impair survival signals through cytokine recep-tors can also lead to excessive cell death, resulting in SCID. The

common cytokine receptor -chain ( c) is a component of the recep-tors for different cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) [129]. Their actions are simultaneously inactivated both in X-linked SCID [33,130], which results from mutations in the gene encoding c, and in JAK3-deficient SCID [35,131], because JAK3 associates with c. In humans, these forms of SCID are character-ized by a profound reduction of T cells but normal or increased B cell number [132]. T cell development also fails to occur in the absence of the chain of the interleukin-7 receptor (IL-7R ) [133]. Based on the association of c with IL-7R and the similar pheno-type caused by these deficiencies, SCID caused by c or Jak3-deficiency is thought to be the result of a requirement for IL-7 in lymphocyte development and homeostasis. The mechanism by which IL-7 performs these developmental and homeostatic regula-tions is uncertain, but the ability of IL-7 to promote expression of Bcl-2 and cell survival has been suggested to be a critical aspect of IL-7 activity. In fact, transgenic expression of Bcl-2 can partially rescue T cell development in IL-7R -deficient mice [134]. More recently, Rathmell et al [135] also demonstrated that the addition of IL-7 to tissue culture supernatants protects purified T cells from both death by neglect and cellular atrophy. This effect was not unique to IL-7, but also occurred with c cytokines IL-2, IL-4 and IL-15 as well as the gp130 cytokine IL-6. It has been suggested that the trophic effect of IL-7 is not caused by forcing T cells to enter the cell cycle but is rather the result of preventing the decline in glycolytic and respiratory rates that occurs in neglected cells.

Fig. (2). Pathway of pyrimethamine-induced apoptosis in activated periph-

eral blood lymphocytes. Activated lymphocytes are vulnerable to the

“physiological” signal represented by the triggering of CD95/Fas with the

consequent activation of caspases 8 and 10. Similarly, pyrimethamine in-

duces apoptosis via a mechanism bringing into play the upstream caspases 8

and 10, although it does not require CD95/Fas engagement. Pyrimethamine-

mediated apoptosis is significantly inhibited by caspase-8 (Z-IETD-fmk), -

10 (Z-AEVD-fmk), and -3 (Z-DEVD-fmk) inhibitors. A direct activity on

mitochondria can also be taken into account. In fact, pyrimethamine leads to

the loss of mitochondrial membrane polarization ( ) and cytochrome c

release from mitochondria, that are considered hallmarks of apoptosis-

associated mitochondrial modification. In addition, the expression of BCL-

2, a negative regulator of mitochondrial apoptotic activity, is down-

regulated by pyrimethamine.


Common Variable Immunodeficiency (CVID)

CVID is an heterogeneous disorder characterized by impaired secretion of immunoglobulins, reduced antibody production, and recurrent infections. Subgroups of patients have increased risks for splenomegaly, granulomatous lesions, autoimmune diseases and malignancies [136]. Despite intense investigation, the aetiology as well as the pathogenesis of CVID, years after its initial recognition, remain largely unknown. Recently, it has been demonstrated that mutations of ICOS [137], TACI [138, 139] and CD19 [140] can result in CVID, although these defects account for only 5 to 10 % of the cases. A major obstacle in characterizing the molecular events leading to CVID is certainly the great heterogeneity of the disease. However, increasing data suggest that there is an underly-ing T cell disorder in most cases. T cells of CVID patients display several features that can be mimicked by a prolonged stimulation in vitro, including the predominance of a memory phenotype, an up-regulation of activation markers, a restriction of TCR variable (BV) chain repertoires, and an increased apoptosis [141-148]. Various reports suggest that the increased susceptibility to apopto-sis, seen in lymphocytes from CVID patients, could be one of the mechanisms implicated in the significant lymphopenia present in CVID and mainly affecting the CD4

+ naïve subset [142, 143, 145,

148]. A positive role of the Fas/FasL system in the increased T cell apoptosis has been suggested by some studies [142, 143, 148]. However, in one study [145] no differences between Fas and FasL expression on CVID and normal T cells were detected. These con-tradictory results may possibly be due to the different phenotypic and clinical characteristics of the patients studied. Although T cells from CVID patients show a persistent immune activation in vivo, attempts to induce Fas-mediated apoptosis, in the presence or ab-sence of activation with mitogenic stimuli, were unsuccessful [143,149]. The impossibility of inducing Fas-mediated apoptosis was hypothesized to be related to the increased serum levels of soluble Fas resulting in the neutralization of the antibody to Fas and/or to the prominent type 1 cytokine profile of CVID patients which was shown to have a protective role in preventing apoptosis [143].

Although most CVID patients show reduced IL-2 production [150], which plays an important role in preventing cell death up-regulating antiapoptotic Bcl-2 family proteins [151], no abnormali-ties in Bcl-2, bax and bcl-xl expression or in the bax/Bcl-2 ratio were demonstrated in both CD4

+ and CD8

+ CVID T cells [143,

145]. Defects of the CD28 receptor pathway, leading to IL-2 depri-vation, have been related, by Di Renzo et al [144], to increased apoptosis in CVID. Costimulation of the CD28 receptor which normally provides, following engagement of the TCR/CD3 recep-tor, a second signal necessary for IL-2 secretion, in CVID did not significantly increase IL-2 production, [152]. Several reports have also shown an activation of the TNF- system in CVID [153, 154]. TNF- is a well known inducer of apoptosis which exerts its effects by binding to two cell surface receptors: TNF-RI and TNF-RII [155]. Di Renzo et al. [145] showed that both TNF-RI and TNF-RII mRNA expressions were significantly increased on CVID CD4

+ T

cells and suggested that this overexpression could contribute to the accelerated T cell death enhancing T cell suceptibility to increased and/or normal levels of circulating TNF- . Increased serum level of TNF- accompanied by glutathione depletion in CD4

+ lympho-

cytes, particularly in the naive subset, have also been reported by Aukrust et al. [156]. Defective glutathione levels were more pro-nounced in those CVID patients characterized by chronic immune activation, low numbers of CD4

+ lymphocytes in peripheral blood

(< 400 cells/μl) and splenomegaly. It has been found that TNF- stimulation of T cell lines and endothelial cells decreases total glu-tathione levels in these cells by causing enhanced ROS generation, which in turn leads to the consumption of reduced glutathione [157, 158]. Thus, increased TNF- activation, may, at least, be responsi-ble for the glutathione depletion and increased levels of oxidative

stress [156]. On the other hand, increased oxidative stress has been related to enhanced apoptosis [159].

Recently, we performed in 60 CVID patients a wide panel of immunological investigations aiming specifically at a quantitative and qualitative evaluation of the T cell compartment [148]. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which reflected in a parallel loss of CD4

+ naive T cells.

A strong correlation between the number of CD4+ naive T cells and

clinical features was observed, supporting the subgrouping of pa-tients according to their number of naive CD4

+ T lymphocytes. A

reduced thymic output and disrupted CD4+ and CD8

+ TCR reper-

toires paralleled the contraction of CD4+ naive T cell pools. The

evaluation of activation markers (i.e. CD95 and HLA-DR mole-cules) and cytokine production (IFN- ) indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis.

In our opinion, CVID represents a model of primary immu-nodeficiency in which, similarly to that observed in chronic viral infections as HIV-infection, prolonged T cell activation, followed by an increased apoptosis results in a progressive exhaustion of the renewal capability of the thymus. Attempting to maintain a normal cell number, peripheral naive T cells intensely proliferate, but to the detriment of the diversity of T cell repertoires. A restricted TCR repertoire might result, in turn, in a decreased ability to counteract infections or cancer. The profound reduction of the CD4 subset, detected in CVID by our and other groups, as compared to the CD8 one, may be due to differences in their regenerative pathway [160]. Differently from the reconstitution of CD8

+ T cells which is rapid

and occurring by peripheral expansion, that of CD4+ T cells is

slower and depends on thymic output [161,162]. Furthermore, the regenerative capability of the CD4

+ T cell compartment is also re-

duced by the proneness of CD4+ T cells to undergo apoptosis

[163,164].

Current therapy for CVID includes regular infusions of intrave-nous immunoglobulin and antibiotics as needed, but significant morbidity and mortality remain [136]. Therapeutic strategies capa-ble of hindering apoptosis of T cells could help in reversing the unbalanced lymphocyte homeostasis, thus leading to an ameliora-tion of clinical status. With regard to this, Cunningham-Rundles et al demonstrated that IL-2 therapy was able to enhance T cell func-tions and reverse T cell anergy in most CVID patients [165-167]. They suggested that CVID patients who might be likely to benefit from this therapy are those patients with defective T helper function as a central abnormality, in which treatment with IL-2 may augment antibody responses as well.

Chromosome 22q11.2 Deletion Syndrome

Chromosome 22q11.2 deletion syndrome (DiGeorge syn-drome/velocardiofacial syndrome) is a disorder classically associ-ated with congenital cardiac defects, immune deficiencies secon-dary to aplasia or hypoplasia of the thymus, and hypocalcemia due to small or absent parathyroid glands [168]. The thymic hypoplasia is seen in more than 80% of the patients with the deletion regardless of the other clinical manifestations of the syndrome. The spectrum of immunodeficiency ranges from absent T cells due to thymic aplasia to normal or near-normal T cell numbers [169-174]. In gen-eral, the T cell deficit improves with age, and many patients pro-duce normal numbers of T cells by the end of the first year of life. T cell function is typically preserved, although standard mitogen pro-liferation assays are usually diminished when the T cell count is very low, corresponding to the diminished numbers of cells compe-tent to respond to the stimulus [171]. Immunoglobulin A (IgA) deficiency, hypogammaglobulinemia, and defects in functional antibody production have occasionally been described and are thought to be secondary to the T cell defect [175-179]. The immu-nodeficiency arises as a consequence of the thymic hypoplasia;


residual thymic tissue is comprised of normal thymic components and the orderly sequence of maturation appears to be preserved allowing T cell differentiation [168]. No significantly altered ex-pression of Fas, Bcl-2 and p53 was also detected in hypoplastic thymus [180]. However, our group observed a reduction of both spontaneous and CD3- or Fas-mediated apoptosis in lymphoid cells from thymic tissue of two patients with chromosome 22q11.2 dele-tion syndrome as compared in comparison with a normal thymus [181]. Differently, in peripheral blood Gupta et al [182] observed high levels of apoptosis of both CD4

+ and CD8

+ T cells and in-

creased Fas and FasL expression, suggesting that spontaneous apoptosis in peripheral T lymphocytes, at least in part, may be re-sponsible for T cell deficiency, mainly affecting the CD4 subset. The predominance of a memory phenotype, a peripheral homeo-static proliferation, and a restriction of both CD4

+ and CD8

+

TCRBV repertoires have been demonstrated by our and other groups [183,184]. Also in this case, the increased activation occur-ring in vivo could be predominantly responsible for the enhanced apoptosis.

APOPTOTIC MECHANISMS IN AUTOIMMUNITY DE-

VELOPMENT

Mechanisms which control apoptosis induction and regulation play a key role in the balance between cell proliferation and death [91]. Imbalance of these mechanisms may contribute to the devel-opment of autoimmune diseases as defective apoptosis of immune cells leads to dysregulated autoreactive cell proliferation [109,185].

Several factors are involved in the induction of resistance to apoptosis as follows: 1) interactions among soluble or membrane molecules and their ligands (i.e. Fas/Fas-ligand, CD40/CD40-ligand, CD80(B7.1)-CD86(B7.2)/CTLA-4; TNF-alfa/TNF-R); 2) the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL and “in-hibitory receptors” like CD94/NKG2A and KIR2D); 3) modifica-tions of death-inducing signal complex (DISC) proetins like FADD, TRADD and RAIDD; 4) defects in caspases activation [186-190]. In the present chapter we briefly summarize recent researches deal-ing with the dysregulation of apoptotic mechanisms in systemic and organ-specific autoimmune diseases.

Apoptosis in Rheumatoid Arthritis

In rheumatoid arthritis circulating inflammatory cells and lo-cally proliferating fibroblasts, lymphocytes and endothelial cells infiltrate the inflamed synovium. The dysregulated cell proliferation is attributable to defects in apoptosis activation which are mediated by cytokines, immunecomplexes, toll-like receptors ligands and hormones (estrogens and their metabolites) detectable in the micro-environment as well as by CD40/CD40L cell-cell interactions [191,192]. In monocytes/macrophages of rheumatoid synovium Fas-dependent apoptotic pathways are blocked by FLIP, which inhibits caspase 8 activation, whereas NF-kB and PI3K/AKT-1 are activated leading to Bcl-2 upregulation, mitochondrial membrane stabilization and apoptosis inhibition. The inhibitors of procaspase 3-caspase 3 pathway X-IAP and p21/Waf-1are also involved in the dysregulation of monocyte/macrophage survival [193-195]. Ep-stein-Barr virus (EBV) may infect neutrophils and macrophages triggering their apoptotic death and decreasing their phagocytic activity. EBV infection stimulates TNF-alpha secretion which in-creases CD34+ cells apoptosis but decreases B cell apoptosis and the development of autoreactive cells [196-198]. Several autoanti-bodies are detectable in rheumatoid arthritis serum including anti-fillagrin antibodies. Filaggrin is a protein involved in the aggrega-tion of cytokeratin filaments and is synthesized in epithelial cells as a phosphorylated precursor. During differentiation of epithelial cells, the precursor is dephosphorylated, cleaved and finally deimi-nated. Anti-fillagrin antibodies do not react with the precursor molecule but only with deiminated filaggrin. Deimination is a post translational modification involving the conversion of arginine

residues into citrulline residues. The enzyme responsible for this modification, peptidylarginine deiminase (PAD), has been detected in a wide number of mammalian tissues and four isoforms of the enzyme have been identified. PAD V isoform has been localized in precursors of macrophages and neutrophils found in inflamed sino-via. PAD is a Ca-dependent enzyme inactive at normal intracellular Ca concentrations. Calcium ionophores that trigger apoptosis of various cells also activate the enzyme and induce protein deimina-tion [199].

Apoptosis in Systemic Autoimmunity: Connective Tissue Dis-eases

In systemic sclerosis (scleroderma), although the number of cutaneous fibroblasts is in the normal range their rate of collagen production is strongly increased under the influence of cytokines produced by leukocytes that infiltrate the skin and of the autocrine production of soluble factors including transforming growth factor-beta (TGF-beta) [200]. The signal transduction program down-stream TGF-beta is activated, as a result of elevated levels of its receptor and/or of the deregulation of the Smad3 factor which is responsible of the signals induced by TGF-beta and controls the ability of TGF-beta to induce fibrosis in vivo [201]. The acquired resistance to programmed cell death of the sclerodermic fibroblasts may contribute to the maintenance of the signalling and of the acti-vated phenotype. In parallel, the recognition of apoptotic cells in-duces per se the production of TGF-beta by cells of the innate im-mune system [202]. Defects in the clearance of apoptotic cells may contribute to the autoantigen spreading and to chronic inflammation and to immune-mediated tissue damage

Apoptotic phenomena play a key role also in the development of systemic lupus erythematosus (SLE). An increased production of apoptotic bodies along with a strong reduction of serum DNase activity which is involved in their clearance has been detected in SLE patients. However, a detailed analysis of DNase gene mutations in SLE is lacking and it is unknown whether these mutations may represent a diagnostic marker for SLE development. Moreover, phagocytosis of anti- GPI-antibody coated apoptotic cells by dendritic cells, followed by the presentation of their antigenic peptides to autoreactive T lymphocytes, may contribute to the maintainance of autoimmune responses in SLE [203,204]. At present, it is not known if these new autoantigens can be utilized as diagnostic tools in SLE patients.

Apoptosis and Organ-Specific Autoimmunity

As far as organ-specific autoimmune diseases are concerned, dysregulation of Fas and Fas-L expression on thyroid cells and in infiltrating lymphocytes has been reported. Moreover, the associa-tion between Graves’ disease and CTLA-4 polymorphisms has been described and it has been hypothesized that CTLA-4 alleles may contribute to the severity of thyrotoxicosis, the development of ophthalmopathy, the risk of relapse following anti-thyroid drugs and the presence other co-existing autoimmune disorders [205]. Finally, elevated serum levels of soluble CTLA-4 (sCTLA-4) have been detected in patients with autoimmune thyroid diseases and it has been proposed that sCTLA-4 may compete with membrane CTLA-4 for CD80/CD86 binding leading to a decrease of the in-hibitory signal [206]. These findings suggest the existence of poten-tial correlations between autoimmune and lymphoproliferative dis-eases also taking into account the elevated risk for the development of low grade lymphomas in patients with coeliac disease and Sjögren syndrome.

APOPTOSIS IN GASTROINTESTINAL IMMUNO-

MEDIATED DISEASES

In the GI tract, as well as all other organs, the balance between cell proliferation and cell death plays a critical role in local physiol-ogy and pathology. Cell death is regulated by a highly controlled


process, known as apoptosis, which also modulates immune and inflammatory responses by inducing leukocyte activation and sur-vival, limiting clonal expansion and cytokine production. Insuffi-cient growth factor production or deprivation induces T cell apopto-sis and the entire mechanism is regulated by a fine balance of dif-ferent molecules acting as anti- and pro-apoptotic factors. As a consequence, diseases may be induced by either a reduction or an increase of apoptosis. Among GI diseases with an immuno-mediated pathogenesis, IBD such as CrD and UC are examples of diseases induced by abnormally decreased apoptosis. On the other hand, increased apoptosis has a role in the pathogenesis of coeliac disease and associated diseases.

Direct and Indirect Mechanisms Leading to Decreased Apopto-sis are Associated with the Pathogenesis of IBD

Apoptosis plays a crucial role in IBD. Immune responses in the mucosa are frequently characterized by major expansions of anti-gen-specific T cells that have potent effector functions. Although this is important for host defense, it might also lead to effector cell populations with substantial autoreactivity and the potential to cause mucosal inflammation. The mucosal immune system has developed several strategies for the control of mucosal immune responses. Among these is the regulation of apoptosis that either occurs via an active mechanism involving death receptors such as Fas or TNFR stimulation or via a passive mechanism such as fol-lowing lymphokine withdrawal.

Direct (Fas-Mediated) Apoptosis

Most LP CD4 T cells express memory cell markers (CD45R0) and constitutively high levels of Fas. In contrast to LP T cells from control patients, those from patients with IBD exhibit defective Fas-induced apoptosis upon stimulation via CD2. This led to the sug-gestion that lamina propria T cells are in a partially anergic state. This defect in inflamed CrD tissue is accompanied by elevated Bcl-2 levels [207]. In addition, normal levels of Fas and Fas ligand are expressed thus suggesting that failure of CrD LP cells to respond to Fas induced apoptosis may be secondary to changes in the expres-sion and/or activity of intracellular molecules which participate in Fas mediated programmed cell death. For instance, Flip (Flice in-hibitor protein), acts conferring resistance to Fas mediated apopto-sis of both CrD and UC lamina propria lymphocytes [208]. Al-though LP T cells in IBD express the same amount of Fas on their surface, they are less sensitive to Fas-mediated apoptosis than con-trol cells. However, resistance of LP T cells against apoptosis in CrD is not restricted to the Fas–FasL system, as recent data show that CrD T cells grow more rapidly in response to IL-2 than do control cells and are significantly more resistant to apoptosis in-duced by IL-2 deprivation. These abnormalities are associated with an imbalance of Bcl-2 positive/ Bax positive mucosal cells. Insuffi-cient T cell apoptosis may interfere with clonal deletion, mainte-nance of tolerance and result in inappropriate T cell accumulation contributing to chronic inflammation [209].

Moreover, recent studies have shown that decoy receptors DcR1, DcR2 and DcR3, that compete with CD95 are highly ex-pressed in T cells from inflamed mucosa in UC patients [210].

In addition, apoptosis related genes such as Bcl-2 antago-nist/killer, B lymphoid tyrosine kinase, caspase 14, Harakiri (a proapoptotic member of the Bcl-2 family of proteins), TNF receptor 2, and TRAF1, were highly expressed in epithelial cells isolated from patients with UC [211].

This broad resistance of T cells to apoptosis involving mito-chondrial activity in inflamed tissue occurs because of increased levels of Bcl-2 [212]. Overall, the picture that emerges is that, in the uninflamed gut, T cells manifest increased spontaneous or activa-tion-induced apoptosis mediated by the Fas pathway that places a limit on the expansion of T cells following direct and bystander

stimulation by specific antigen. By contrast, in inflamed tissue, T cells resistant to apoptosis, exhibit prolonged survival and increased inflammatory cytokines production [213].

Indirect (Cytokine-Mediated) Apoptosis

An important role in regulation of apoptosis has been related to cytokines which can stimulate or down-regulate apoptotic mecha-nisms.

IL-23 is essential for T cell-mediated colitis and promotes in-flammation via IL-7 and IL-6 [214]. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifi-cally activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intes-tinal inflammation. IL-23 shares the same p40 subunit with IL-12 and anti-p40 antibodies neutralize both IL-12 and IL-23, reversing intestinal inflammation in both humans and animal models [214].

It has been reported that IL-10 is important in maintaining im-mune homeostasis in the gut, demonstrated by the fact that IL-10 knockout (KO) mice spontaneously develop colitis [215]. In pa-tients with CrD, a decreased IL-10 concentration in the ileum pre-dicted a higher risk of disease recurrence [216]. Systemic IL-10 treatment or IL-10 gene transfer has been successfully used in sev-eral animal models of inflammatory bowel disease, such as IL-10 KO mice and severe combined immunodeficiency transfer colitis [217].

A pathway of T cell activation driven by IL-6/IL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CrD [212]. Novel therapeutic strategies aim at restor-ing mucosal T cell susceptibility to apoptosis through targeting of signal transduction pathways that are crucial for augmented resis-tance of T lymphocytes against apoptosis. For instance, a newly developed humanized anti-IL-6R monoclonal antibody that induces intestinal T cell apoptosis showed clinical efficacy in patients with active CrD [218].

IL-10 can block apoptosis of intestinal epithelial cells by inhib-iting COX-2 production [219]. In keeping with these data gene polymorphisms associated with reduced production of IL-10 have been reported as risk factors for developing IBD [220]. Also inhibi-tors of IL-23 and IL-12 are able to reverse colitis [221] and anti-interleukin 12 treatment induces Fas mediated apoptosis of Th1 cells in experimentally induced colitis in mice [222].

Increased Apoptosis is Associated with the Pathogenesis of Coeliac Disease

In contrast to IBD, where the pathogenesis has been associated to a failure in feedback apoptotic mechanisms, as discussed above, increased apoptosis may be linked to the pathogenesis of Coeliac disease (OMIM 212750). This is a gluten-dependent entheropathy occurring in genetically predisposed individuals and resulting from inflammatory injury to the mucosa of small intestine associated to diverse clinical manifestations whose frequency has been reported to be very high in Caucasians. Coeliac disease is associated with a variety of other diseases which share common genetic susceptibility and pathogenetic mechanisms. For instance, dermatitis Herpeti-formis (DH) is very often associated with coeliac disease. In DH, dermal Bax and Bcl-2 proteins are increased in the dermal perivas-cular compartment and apoptosis plays a major role in the patho-genesis of cutaneous lesions [223]. Development of coeliac disease is strongly linked to genetic factors both HLA related [224] and non-HLA related [225]. The effector phases of small bowel tissue destruction in coeliac disease appear to be mediated by cytotoxic T and NK cells through both apoptosis mediated tissue damage and release of proteolytic enzymes such as perforins. Apoptosis is me-diated through ligation of FasL expressed on gliadin-activated T-cells.


In untreated coeliac disease, FasL and perforin expression by IEL is increased, and correlates with the level of enterocyte apopto-sis [226]. This phenomenon also occurs at the peripheral level and is possibly responsible for lymphopenia, exposure of membrane phospholipids and subsequent production of autoantibodies [227].

IEL of CD3 negative cells expressing NK markers such as CD16 and CD161 are found in coeliac disease [228] and at the molecular level both the absolute number and the percentage of lymphocytes expressing perforin mRNA are increased [229]. This expanded NK population may account for the increased levels of perforin in the gut.

Cytokines are also involved in coeliac disease activation-induced cell death. Epithelium-derived IL-15 signalling via IL-15R is critical for the development, activation and survival of IEL [230]. IL-15 can also induce CD94 positive IEL to gamma inter-feron production and FasL-mediated cytotoxicity [231].

These data, although presently limited to biological models, may soon became useful for the clinical benefits of patients. In fact, blocking IL-15 may suppress uncontrolled IEL activation and sur-vival, with the potential to prevent both tissue damage and lym-phomagenesis in coeliac disease [230].

CONCLUSION

In conclusion, the interactions among soluble and membrane molecules as well as the balance between pro-apoptotic and anti-apoptotic factors, both in effector and target cells, seem to represent key mechanisms underlying the development of autoimmune dis-eases. Although immune tolerance seems to be achieved mainly through central selection implying apoptotic mechanisms, some of which have been described in the chapter on thymocyte differentia-tion, this process is by far insufficient to delete autoreactive clones, which may survive in the periphery. If normal regulatory systems fail or homeostatic peripheral expansion, a physiologically normal control of lymphopenia, is a recurrent phenomenon, selective ad-vantage for autoimmune T cell growth occurs. Immunodeficiency states are often characterized by the paradoxical occurrence of auto-immunity, so that a more detailed characterization of these interac-tions is required in order to better define the pathophysiology of autoimmune diseases. Finally, the possibility to interfere with regu-latory mechanisms of apoptosis, aiming either to block or to en-hance it at different time points, represents a promising approach for the development of new immunomodulatory therapies.

ACKNOWLEDGMENTS

Studies reported in this paper have been supported by grants Ricerche di Facoltà 2007 to R.P.

ABBREVIATIONS

AIRE = Autoimmune regulator

ALD = Autoimmune lymphoproliferative disease

ALPS = Autoimmune lymphoproliferative syndrome

APC = Antigen presenting cell

GPI = 2 Glycoprotein-I

COX-2 = Ciclooxygenase-2

CrD = Crohn’s disease

CVID = Common Variable Immunodeficiency

DC = Dendritic cell

DH = Dermatitis herpetiformis

DISC = Death-inducing signal complex

DN = Double negative

DP = Double CD4+CD8+ positive

EBV = Epstein-Barr virus

ETP = Early thymic precursor

FADD = Fas associated death domain

FasL = Fas ligand

FLIP = FLICE inhibitory protein

c = Common gamma chain

GI = Gastrointestinal

HIV = Human immunodeficiency virus

HLA = Human leukocyte antigens (histocompatibility complex)

IBD = Inflammatory bowel disease

ICAM-1 = Intercellular adhesion molecule-1

ICOS = Inducible co-stimulator

IEL = Intraepithelial lymphocytes

IFN = Interferon

IL = Interleukin-

ISP = Immature single positive

JAK = Janus kinase

KO = knoçck out

LP = Lamina propria

MCL-1 = Myeloid cell leukemia sequence 1

MHC = Major histocompatibility complex

NFAT = Nuclear factor of activated T cells

NF- B = Nuclear factor - B

NK = Natural killer

PAD = Peptidylarginine deiminase

SCID = Severe combined immunodeficiency

SLE = Systemic lupus erythematosus

TACI = Transmembrane activator and CAML interactor

TCR = T cell receptor

TEC = Thymic epithelial cells

TGF = Transforming growth factor-beta

TNF = Tumor necrosis factor

TP = Thymic precursor

TRAIL = TNF-related apoptosis-inducing ligand

Treg = T regulatory cells

TSA = Tissue specific antigens

UC = Ulcerative colitis

VCAM-1 = Vascular cell adhesion molecule-1

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Apoptosis in the Homeostasis of the Immune System and in Human Immune Mediated Diseases

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