Apoptosis in immunity

In the immune system, apoptosis is used as a means to dispose of unwanted cells time and again. In many cases the CD95 molecule (Fas/APO-1), a member of the TNF/NGF receptor family, is used to transduce the death signal. Some of the situations where

Proapoptotic signals

Convergence point

Convergence point

CrmA p35

Cleavage of multiple substrates

Phenotypic changes characteristic of apoptosis

Figure 5 Molecular events in apoptosis. Members of the cas-pase (ICE/CED-3) protease family are depicted as being at a point just below the convergence point for diverse proapoptotic stimuli. Activation of ICE/CED-3 family proteases may lead directly to the apoptotic phenotype through cleavage of specific substrates, although this is speculative. From current evidence, some apoptosis-repressor proteins (Bcl-2) appear to act upstream of the point of activation of at least some of the members of the ICE/CED-3 family, while others (CrmA, p35) act directly as inhibitors of these proteases.

apoptosis plays a key role in immunity are described below.

Thymic selection

It has been estimated that approximately 95-98% of thymocytes that enter the thymus are eliminated there. Thymocytes die at several stages of the maturation process; either because they fail to rearrange their TCR genes successfully, because they exhibit strong reactivity with self (negative selection), or because they fail to recognize self-MHC at all (death by neglect). Studies in the late 1980s and the early 1990s established that thymocytes die in all of these cases due to the induction of apoptosis in these cells. Most of the cell deaths that occur in the thymus do so in the cortex and at the corticomedullary boundary at the double-positive stage (CD4+CD8 + ) of thymocyte development. Upon entry into the medulla, single-positive thymocytes (CD4+CD8~ or CD4~CD8+) become much more resistant to the induction of apoptosis. The precise nature of the signal that provokes death of cortical thymocytes has not been established. The possible involvement of CD95-CD95 ligand interactions is ruled out due to the fact that thymocyte maturation is largely unaffected in mice that carry loss-of-function mutations in CD95 (lpr/lpr) or the CD95 ligand (gld/gld). The thymocyte death signal is similarly unaffected by over-expression of Bcl-2, a 26 kDa protein that blocks many other forms of cell death.

Despite the enormous amount of cell death that goes on in the thymus, it is remarkable how few apoptotic cells are actually detectable in tissue sections of this organ. This puzzle can be explained if it is recalled that apoptotic cells are readily recognized by cells with phagocytic capability - by virtue of the membrane changes on these cells - and are eaten before they exhibit the morphologic features of apoptosis. Recent studies have implicated resident F4/80~ macrophages as the effectors of this efficient clearance process for thymocytes undergoing negative selection, whereas F4/80+ macrophages appear to remove thymocytes that have died by neglect.

Peripheral deletion

The small minority of thymocytes that are positively selected exit the thymus and migrate to the peripheral lymphoid organs. Here they are faced with the possibility of reacting with previously unencountered self-antigens. Should this happen, the autoreactive mature T cell, all going to plan, should then undergo apoptosis, probably as a result of receiving an inappropriate activation signal (such as TCR engagement in the absence of CD28 costimulation). Should this peripheral deletion process fail for any reason, then autoimmunity can result. Unlike in the thymus, CD95-CD95L interactions do appear to be important for peripheral deletion, as both lpr and gld mice are impaired in peripheral deletion and develop a systemic lupus erythematosus (SLE)-like autoimmune disease as a result.

Cleavage of multiple substrates

Peripheral T and B lymphocytes are also eliminated after an immune response, otherwise the numbers of these cells would steadily increase after each successive encounter with antigen. While such an increase does happen to some extent (i.e. precursor frequencies to recall antigens are higher than to previously unencountered antigens), clearly the majority of clonally expanded lymphocytes do not survive long after an immune response has ended. Studies in recent years have established that these excess lymphocytes die by apoptosis. Once again, CD95-CD95L interactions have been implicated in this process. While mature peripheral T cells constitutively express CD95 on their surface, they are largely resistant to CD95L-mediated killing until they are activated, whereupon they now acquire sensitivity to CD95 ligation. Thus, activated T cells die upon encounter with CD95L-bearing cells. Since the expression of CD95L is strictly restricted to certain tissues, it is not clear what cells do the actual killing. It may be the activated T cells themselves, since CD95L is upregulated on lymphocytes upon activation, or a subset of dendritic cells or other antigen-presenting cells.

CTL and NK killing

Cytotoxic lymphocytes (CTLs and natural killer (NK) cells) are capable of killing target cells via two distinct pathways: by delivering cytotoxic granules on to the plasma membrane of the target cell, or by cross-linking the CD95 molecule on the target cell surface via CD95 ligand. In both cases, the end-result is the activation of the endogenous death machinery in the target cell, resulting in death from within - a kind of assisted suicide.

Granule-dependent killing is thought to be the major mechanism of cytotoxic lymphocyte killing. Cytotoxic cell granules contain perforin, a pore-forming protein similar to the C9 component of complement, as well as a series of enzymes - several of which are serine proteases - called granzymes. Perforin is thought to act as a conduit or channel to allow the entry of granzymes into the target cell cytoplasm. Of the seven or so granzymes that have been characterized to date, only one of these, granzyme B, has been shown to be both necessary and sufficient, in combination with perforin, to induce apoptosis of target cells. Granzyme B is an unusual protease in that it cleaves after Asp residues, a property that it shares with the caspase family of cysteine proteases discussed earlier. Recent studies have shed light on the mechanism whereby granzyme B activates the endogenous death machinery in the target cell. A number of groups have found that granzyme B is capable of directly cleaving and activating several members of the caspase family, but not ICE itself. Thus, granule-dependent killing operates by directly activating the endogenous cell death machinery in the target cell by processing members of the caspase family to their active forms.

The other mechanism of cytotoxic lymphocyte killing involves a CD95 ligand-bearing cell interacting with, thereby cross-linking, CD95 on the target cell surface. As mentioned above, CD95 is a member of the TNF/NGF receptor family that transduces, upon trimerization, a death signal into cells bearing this receptor. CD95 was originally identified in 1989 by two separate groups and is commonly called two different names as a result: Fas or APO-1. The molecular mediators of the CD95-associated death signal have recently begun to emerge. Briefly, the cytoplasmic tail of CD95 contains a stretch of amino acids that are critical for the CD95-mediated death signal (the death domain), but this region does not have any obvious signaling function (such as kinase or phosphatase activity). Other death domain-containing proteins have since been discovered, such as FADD (Fas-associated death domain), that can associate with the CD95 death domain, thereby propagating the apoptotic signal. Thus, the death domain appears to be a protein-protein interaction domain. FADD then recruits caspase-8 (MACH-1/FLICE) which becomes processed to its active form, thus propagating the death signal.

Maintenance of immune privilege

It has been known for over a hundred years that certain sites in the body enjoy immune privilege. That is to say, immune responses do not generally occur at these sites. Classically, an immune-privileged site is defined as an area where introduction of allogeneic or xenogeneic cells does not result in immediate rejection as it would in other areas of the body. This definition has subsequently been broadened to include infectious organisms and tumors. Examples of immune-privileged sites include the eye, the testis and the central nervous system - all of which arc tissues where the benefits of an immune response are outweighed by the dangerous consequences of a vigorous immune reaction in such delicate and vital tissues. While it was initially thought that immune privilege was maintained by physical barriers that contained antigen and prevented infiltration of the site by myeloid cells and lymphocytes, this is now known to be untrue because these cells do enter privileged sites but are somehow inactivated. Recent studies on the eye and the testes appear to have solved this puzzle. In these organs, it transpires that CD95 ligand is abundantly expressed such that infiltrating lymphocytes and neutrophils - both of which express surface CD95 - are induced to undergo apoptosis upon entry into these sites. As elegant proof of this, mice carrying a non-functional CD95-ligand (gld) do not enjoy immune privilege and, in the case of the eye, display marked infiltration upon introduction of a virus (herpes simplex virus type 1, HSV-1) into the anterior chamber of this organ -resulting in keratitis. In stark contrast, introduction of HSV-1 into the eye of wild-type mice results in death of infiltrating lymphocytes and neutrophils and containment of the infiltrating cells to the anterior chamber. Similarly elegant transplantation experiments of testes from lpr and gld mice yielded a similar conclusion that CD95 ligand was responsible for maintaining immune privilege in this organ also. Other studies have demonstrated that CD95L is also abundantly expressed in other immune-privileged sites such as the brain, ovary, uterus, adrenal gland and prostate, suggesting that a similar protective mechanism from immune responses operates in these tissues also.

B cell development and affinity maturation

Like T lymphocytes, the majority of newly formed B lymphocytes fail to migrate to the peripheral lymphoid organs due to the induction of apoptosis in these cells. B cells are produced throughout life in the bone marrow but only a small minority of these cells, as is the case with thymocytes, will be allowed to enter the peripheral blood. Most developing B-lineage cells die by apoptosis, either because of aberrant immunoglobulin (Ig) gene rearrangements or due to encounter with high concentrations of high-affinity antigens (akin to negative selection). It has been estimated that approximately 75% of developing B cells die in the bone marrow at the transition between large pro-B to small pre-B cell stage. The small fraction of virgin B cells that exit the bone marrow travel to the spleen and then on to the lymph nodes. In the peripheral lymphoid tissues B cells fall into two compartments, short-lived (2-3 days) and long-lived (several weeks). Short-lived B cells probably die (by apoptosis) through failure to encounter antigen and are rapidly replaced by newly formed B cells from the bone marrow. B cells that receive a survival signal (antigenic stimulation) likely switch to the long-lived state, although there has been some debate concerning whether short-lived and long-lived B cells are derived from separate bone marrow precursors or a common progenitor. In either case, long-term survival of B cells appears to require repeated re-stimulation with antigen. Long-term B cell survival appears to be regulated, at least in part, by expression of the Bcl-2 protein. Bcl-2 transgenic mice display elevated peripheral B cell numbers and extended B cell memory.

B cells that manage to persist also run the gauntlet of apoptosis during affinity maturation in the germinal centers. During the germinal center reaction, activated B cells acquire point mutations in the V regions of their immunoglobulin genes that may or may not increase their affinity for antigen and can even result in autoreactivity. The majority of B cells that undergo this hypermutation process are eliminated via apoptosis, with mutants that have a high affinity for antigen being rescued from a similar fate due to encounter with their specific antigen in the context of CD40 ligand (gp39) costimulation. CD95 also appears to play a role in this elimination process because activated B cells acquire sensitivity to CD95 unless they receive concurrent engagement of the CD40 molecule. This may also explain why lpr mice (that are defective in CD95) also display a B cell defect and develop and SLE-like autoimmune disease.

Resolution of inflammation

One of the earliest events in an immune response involves the dramatic accumulation of infiltrating neutrophils and other cells of the myeloid lineage at the site of infection. Neutrophils form a very important first line of defense and these cells appear to be well equipped for this task, with a barrage of toxic enzymes and reactive oxygen intermediates within their secondary granules. But what happens to all of these cells when they have completed their task and are no longer required? It is well known that neutrophils have a very short half-life of approximately 12-18 hours. But where do they die and how are they disposed of? By now it will come as no surprise to learn that old unwanted neutrophils die by apoptosis and that damage to cells in the vicinity of dying neutrophils is minimized as they are programmed to die around the time that the wave of macrophage infiltration reaches its peak. Thus, inflammatory macrophages are charged with the task of removing the dying neutrophils and of safely disposing of their potentially destructive contents. The trigger for macrophage recognition of senescent neutrophils, as discussed earlier, appears to be either phosphatidylserine externalization at the surface of the plasma membrane or the appearance of a throm-bospondin-binding moiety on the surface of these cells - depending on whether the phagocytic cells are inflammatory or resting macrophages, respectively. Interestingly, macrophages respond very differently when they phagocytose apoptotic cells, as opposed to yeast particles, for example, as the former do not elicit the release of inflammatory mediators by the macrophage. Whether a distinct phagolysosomal compartment is used for digestion of apoptotic cells has not been explored as yet.

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