Reactive Nitrogen Intermediateinduced Parp Activation In The Immune System

7.1.1 Introduction

Macrophages were among the first cell types identified as a source of nitric oxide (NO) produced by the inducible nitric oxide synthase (iNOS).1-5 NO production represents a major cytotoxic mechanism utilized by macrophages to kill certain pathogens and tumor cells.4-8 Although NO is a free radical capable of inhibiting the respiratory chain,58 inactivating enzymes,910 peroxidating lipids,11-13 and causing cytotoxicity,4,5 cell damage associated with increased NO production is more likely to be mediated by NO-derived oxidants such as peroxynitrite than by NO per se.14 Peroxynitrite (ONOO) is a binary toxin assembled when NO and superoxide (O2) are produced together.14 Peroxynitrite is a very potent inducer of DNA single-strand breaks15-18 and thus poly(ADP-ribose) polymerase (PARP) activation.18-20 Because activated macrophages upregulate iNOS and produce large amounts of NO in parallel with the overproduction of superoxide in a process called "oxidative burst," macrophage activation provides ideal conditions for peroxynitrite formation. Indeed, activated macrophages have been shown to produce ONOO.21 Macrophages and related cell types (e.g., dendritic cells) are present in all lymphoid organs where they sample antigens from the lymph and the bloodstream and present antigens to T lymphocytes. Activated T lymphocytes in turn secrete interferon y (IFNy), which is a potent activator of macrophages. Thus, during antigen presentation cross-activation between antigen-presenting cells (APC) and T cells may result in NO production, peroxynitrite formation, and PARP activation. Indeed, NO production by APCs (Langerhans cells, dendritic cells) and modulation of T-cell function by APC-derived NO has been reported in the literature.22-25 Peroxynitrite-induced PARP activation may occur in the NO-producing cell or in cells localized in the vicinity of the source of NO. We have selected the thymus as a primary lymphoid organ and investigated (1) whether NO is produced in the thymus, (2) whether intrathymic NO production results in peroxynitrite formation in the thymus, and (3) whether PARP activation occurs in thymocytes. Moreover, we have extensively investigated the in vitro effect of peroxynitrite on thymocytes as a model cell type and studied how PARP activation affects the mode of peroxynitrite-induced cell death.

As this chapter focuses on the role of PARP in the reactive nitrogen intermediate (RNI)-induced cytotoxicity, a relatively detailed outline of the role of RNI in the immune system is given. The possible effect of PARP on the formation of T and B

lymphocyte repertoire and some aspects of PARP as a transcriptional regulator of immune functions are also discussed.

7.1.2 Intrathymic Selection Processes

The thymus is a primary lymphoid organ where thymocytes go through a vigorous selection process. During positive selection, thymocytes capable of recognizing self-histocompatibility (MHC) molecules receive a survival signal from cortical epithelial cells, whereas cells unable to bind to self-MHC undergo apoptosis.26-29 Positively selected thymocytes, in turn, migrate deeper into the thymus and make contact with dendritic cells at the corticomedullary junction and in the medulla.27-29 Dendritic cells present self-antigens to thymocytes and induce apoptosis of those carrying T-cell receptors (TCR) with high affinity toward self-antigens (negative selection). As a result of these selection processes, the vast majority of thymocytes (>95%) die within the thymus and only mature T cells with the ability to recognize non-self-antigens in association with self-MHC enter the circulation.27-29 The predominant death signal provided by dendritic cells during negative selection is transduced via the T-cell receptor. However, signaling through the TCR alone does not necessarily lead to apoptosis.28,30,31 For instance, cosignaling via the cell-surface Fas molecule, a member of the TNF receptor family, and through CD28 and Thy-1 surface receptors may also contribute to apoptosis induction.32-36 A role for NO in the negative selection has also been proposed based on findings that in vivo TCR stimulation leads to the upregulation of iNOS in the thymus and to a decrease in the number of CD4+CD8+ thymocytes.3738

7.1.3 Nitric Oxide and Peroxynitrite in the Thymus

We have used NADPH diaphorase (NADPHd) histochemistry to detect NOS activity in the rat thymus. In accordance with previous studies,39,40 we have found numerous NADPHd+ cells in the rat thymus (Figure 7.1) with the highest density observed at the corticomedullary junction.41 Based on the distribution and the morphology (long branching processes) of NADPHd+ cells, they may represent dendritic cells; however, their phenotype has not yet been determined.

We have also detected NADPHd+ corticomedullary cells in the mouse thymus (Figure 7.2) where iNOS positivity of NADPHd+ cells was proved by iNOS immu-nohistochemistry.42 Furthermore, NADPHd+/iNOS+ cells were absent from the thymi of iNOS-deficient thymi proving that NADPHd reaction detects iNOS in our sys-tem.42 We have investigated the inducibility of NADPHd/iNOS in the mouse thymus by using staphylococcal enterotoxin B (SEB), a superantigen that cross-links the MHC molecules of APCs (e.g., macrophages and dendritic cells) with the TCR of thymocytes and T lymphocytes. NADPHd reaction revealed a massive increase in the number of positive cells in the SEB-treated mouse thymus as compared with the naive thymus (see Figure 7.2).42 SEB induced a marked increase in the amount of immunohistochemical staining for iNOS, with a pattern identical to that of NADPHd.42 In the thymi of iNOS-deficient animals, neither NADPHd nor iNOS staining could be detected after SEB challenge.42 The lack of these NADPHd+

FIGURE 7.1 NADPHd+ positive cells in the rat thymus. Thymi from untreated Sprague-Dawley rats were removed, and frozen sections were stained for NADPHd to detect NOS activity. Strongly stained cells with long branching processes were found in the corti-comedullary junction. (Bar: 100 |J,m; original magnification, 100x.)

FIGURE 7.1 NADPHd+ positive cells in the rat thymus. Thymi from untreated Sprague-Dawley rats were removed, and frozen sections were stained for NADPHd to detect NOS activity. Strongly stained cells with long branching processes were found in the corti-comedullary junction. (Bar: 100 |J,m; original magnification, 100x.)

medullary cells in the thymocytes of iNOS knockout mice strongly indicates that the NADPHd staining was, indeed, related to iNOS.

Following the proposal of the participation of iNOS-derived NO in the negative selection, the question arose whether intrathymic NO production by iNOS leads to peroxynitrite formation. For the detection of in vivo peroxynitrite production, we have taken advantage of the fact that unlike NO, peroxynitrite reacts with tyrosine residues of proteins. Therefore, the presence of nitrated proteins in tissues is regarded as a footprint of peroxynitrite and other oxidized NO products.14 43 44 We have detected cells containing nitrated proteins in the thymi of untreated mice (Figure 7.3), indicating the in vivo "basal" production of peroxynitrite in this organ.45 The nitrotyrosine-positive cells were predominantly found in the medulla. In addition, nitrotyrosine-positive cells were found in the thymi of iNOS-deficient mice, although they were less abundant than in the wild-type mice and displayed a predominantly perivascular distribution (Figure 7.3).45

In summary, NO and peroxynitrite are formed at the site of negative selection (at the corticomedullary junction and in the medulla) and these reactive nitrogen intermediates may provide accessory death signals to thymocytes during negative selection.46

7.1.4 Nitric Oxide and Peroxynitrite in Other Lymphoid Organs

Until relatively recently, the thymus appeared to be a unique immune organ where iNOS-derived NO and peroxynitrite are produced without exogenous stimulation.

FIGURE 7.2 NADPHd histochemistry reveals iNOS-expressing cells in the thymus of naive and SEB-stimulated mice. (A) NADPHd+ staining in the thymi of naive mice. (B) Immunohis-tochemical detection of iNOS in the thymi of naive mice. (C, E) NADPHd+ staining in the mouse thymus after SEB stimulation. (D, F) iNOS staining of thymi from SEB-treated mice. (G) NADPHd+ in the thymus of iNOS knockout animals following SEB stimulation. Only endothelial staining could be detected. A similar staining pattern was seen in naive iNOS knockout animals. (H) No iNOS staining could be detected in the thymus of iNOS knockout animals following SEB stimulation. For panels A, B, C, D, G scale bar = 25 |J,m, (original magnification, 400x); for panels E, F, H scale bar = 100 |J,m (original magnification, 100x). (From Virag, L. et al., J. Histochem. Cytochem., 46, 787-791, ©1988. The Histochemical Society. With permission.)

FIGURE 7.2 NADPHd histochemistry reveals iNOS-expressing cells in the thymus of naive and SEB-stimulated mice. (A) NADPHd+ staining in the thymi of naive mice. (B) Immunohis-tochemical detection of iNOS in the thymi of naive mice. (C, E) NADPHd+ staining in the mouse thymus after SEB stimulation. (D, F) iNOS staining of thymi from SEB-treated mice. (G) NADPHd+ in the thymus of iNOS knockout animals following SEB stimulation. Only endothelial staining could be detected. A similar staining pattern was seen in naive iNOS knockout animals. (H) No iNOS staining could be detected in the thymus of iNOS knockout animals following SEB stimulation. For panels A, B, C, D, G scale bar = 25 |J,m, (original magnification, 400x); for panels E, F, H scale bar = 100 |J,m (original magnification, 100x). (From Virag, L. et al., J. Histochem. Cytochem., 46, 787-791, ©1988. The Histochemical Society. With permission.)

FIGURE 7.3 Immunohistochemical detection of nitrotyrosine in the thymus. Cryostat sections of naive wild-type (A) and iNOS knockout (B) mice were stained for nitrotyrosine. Immunoreactive cells were found predominantly in the medulla. A few nitrotyrosine-positive cells of predominantly perivascular localization could also be detected in the thymi of iNOS-deficient mice (B). (Bar = 100 |J,m; original magnification, 100x.) (From Virâg, L. et al., Immunology, 94, 345-355, ©1998. Blackwell Science Ltd. With permission.)

FIGURE 7.3 Immunohistochemical detection of nitrotyrosine in the thymus. Cryostat sections of naive wild-type (A) and iNOS knockout (B) mice were stained for nitrotyrosine. Immunoreactive cells were found predominantly in the medulla. A few nitrotyrosine-positive cells of predominantly perivascular localization could also be detected in the thymi of iNOS-deficient mice (B). (Bar = 100 |J,m; original magnification, 100x.) (From Virâg, L. et al., Immunology, 94, 345-355, ©1998. Blackwell Science Ltd. With permission.)

However, Brito et al.47 have recently demonstrated that nitrotyrosine can be detected in human lymph nodes obtained from routine surgical resections for lung and colon cancers (Figure 7.4). The nitrotyrosine immunostaining was predominantly localized to macrophages; however, lymphocytic and perilymphocytic staining has also been found in the lymph nodes.47

Similarly to our hypothesis for intrathymic iNOS activation,46 Brito et al. have suggested that cross talk between lymphocytes and monocytes/macrophages leads to nitration of proteins in lymphocytes.47 This scenario is supported by experiments where peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes were stimulated with anti-CD3 antibody (T-cell activator) followed by purification of lymphocytes (Figure 7.5). Under these condition, strong tyrosine nitration was detected by Western blotting.47 However, when CD3 stimulation was carried out after the separation of lymphocytes (i.e., in the absence of monocytes), no significant increaese in tyrosine nitration could be detected (Figure 7.5).47

In summary, these results show that activation of the immune system is accompanied by iNOS expression and peroxynitrite formation due to cross-activation of T lymphocytes and monocytes/macrophages. According to this scenario (a similar one to that is depicted in Figure 7.10), activation of T cells by anti-CD3 or superantigens such as SEB results in cytokine (INFy) production by activated T cells. INFy in turn activates monocytes/macrophages/dendritic cells to express iNOS and to switch on the oxidative burst, fueling peroxynitrite production from both sides (NO and superoxide). Peroxynitrite produced by activated monocytes/macrophages triggers tyrosine nitration within the monocytes/macrophages and also diffuses through cell membranes and nitrates tyrosine in lymphocytes.

Peroxynitrite was proposed to inhibit T-cell activation by tyrosine nitration;47 however, a role for PARP cannot be ruled out in this process. The possibility that peroxynitrite produced during immune reactions activates PARP has not yet been investigated. It would be interesting to know whether PARP becomes activated in this process and how PARP activation may affect the activation of both the T cells and cells of the monocyte/macrophage lineage.

FIGURE 7.4 Nitrotyrosine staining in human lymph nodes. Human lymph nodes were obtained from routine surgical resections for lung and colon cancers, and tyrosine nitration was detected by immunohistochemistry. (A) Subcapsular and medullar sinuses and scanty cortical immunoreactivity with antinitrotyrosine. (B) Nitrotyrosine-positive macrophages surrounding subcapsular sinuses. (C) Dilated sinuses containing large numbers of sinus histiocytes with strong cytoplasmic immunolabeling. (D) Multiple parenchymal macrophages with strong granular reactivity and a poorly defined perilymphocyte and lymphocyte labeling with random distribution. (Original magnifications: A: 40x, B and D: 400x, C: 100x.) (From Brito, C. et al., J. Immunol., 162, 3356-3366, ©1999. The American Association of Immunologists. With permission.)

FIGURE 7.4 Nitrotyrosine staining in human lymph nodes. Human lymph nodes were obtained from routine surgical resections for lung and colon cancers, and tyrosine nitration was detected by immunohistochemistry. (A) Subcapsular and medullar sinuses and scanty cortical immunoreactivity with antinitrotyrosine. (B) Nitrotyrosine-positive macrophages surrounding subcapsular sinuses. (C) Dilated sinuses containing large numbers of sinus histiocytes with strong cytoplasmic immunolabeling. (D) Multiple parenchymal macrophages with strong granular reactivity and a poorly defined perilymphocyte and lymphocyte labeling with random distribution. (Original magnifications: A: 40x, B and D: 400x, C: 100x.) (From Brito, C. et al., J. Immunol., 162, 3356-3366, ©1999. The American Association of Immunologists. With permission.)

Another interesting phenomenon is the "burnout" of lymphocytes during sepsis.48 Prolonged sepsis causes a significant decrease in lymphocyte ATP levels, which correlates with decreased lymphocyte proliferative capacity in response to mitogenic stimulation.48 Treatment with ATP-MgCl2 at the onset of sepsis significantly increases lymphocyte ATP levels and proliferative response to mitogenic stimuli.48 Although it has not yet been tested experimentally, PARP appears to be a plausible candidate for being responsible for the lymphocyte burnout. Sepsis is characterized by the overproduction of free radicals, which can activate PARP, and the beneficial effect of ATP supplementation also fits well in this hypothesis.

Free radical/oxidant-induced PARP activation is not the only possible mechanism by which PARP can alter immune activation. Upon recognition of antigen, lymphocytes (both T and B cells) undergo proliferation and express new genes, and B cells differentiate into antibody-secreting plasma cells. All these cellular functions (proliferation,49-51 differentiation,51-59 gene expression60-66) have been found to be regulated by PARP in certain cell types including lymphocytes, macrophages, and granulocytes.

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