commentaries

B-cell lymphomas. N. Engl. J. Med. 359:2313–2323. tance to apoptosis. Clin. Res. 14:4925–4934. lysosomal cell death pathway by oncogene-induced 11. Byrd, J.C., et al. 2006. Select high-risk genetic 15. Cragg, M.S., and Glennie, M.J. 2004. Antibody down-regulation of lysosome-associated mem- features predict earlier progression following specificity controls in vivo effector mechanisms of brane 1 and 2. Cancer Res. 68:6623–6633. chemoimmunotherapy with fludarabine and anti-CD20 reagents. Blood. 103:2738–2743. 20. Davis, T.A., et al. 2004. The radioisotope contrib- rituximab in chronic lymphocytic leukemia: jus- 16. Beers, S.A., et al. 2008. Type II (tositumomab) anti- utes significantly to the activity of radioimmuno- tification for risk-adapted therapy. J. Clin. Oncol. CD20 monoclonal antibody out performs type I therapy. Clin. Cancer Res. 10:7792–7798. 24:437–443. (rituximab-like) reagents in B-cell depletion regardless 21. Horning, S.J., et al. 2005. Efficacy and safety of tos- 12. Johnson, P., and Glennie, M. 2003. The mecha- of complement activation. Blood. 112:4170–4177. itumomab and iodine-131 tositumomab (Bexxar) nisms of action of rituximab in the elimination of 17. Ivanov, A., et al. 2009. Monoclonal antibodies in B-cell lymphoma, progressive after rituximab. tumor cells. Semin. Oncol. 30:3–8. directed to CD20 and HLA-DR can elicit homo- J. Clin. Oncol. 23:712–719. 13. Chan, H.T., et al. 2003. CD20-induced lymphoma typic adhesion followed by lysosome-mediated cell 22. Ostenfeld, M.S., et al. 2008. Anti-cancer agent sir- cell death is independent of both caspases and its death in human lymphoma and leukemia cells. amesine is a lysosomotropic detergent that induces redistribution into triton X-100 insoluble mem- J. Clin. Invest. 119:2143–2159. cytoprotective autophagosome accumulation. brane rafts. Cancer Res. 63:5480–5489. 18. Cerisano, V., et al. 2004. Molecular mechanisms of Autophagy. 4:487–499. 14. Ivanov, A., Krysov, S., Cragg, M.S., and Illidge, T. CD99-induced caspase-independent cell death and 23. Groth-Pedersen, L., Ostenfeld, M., Høyer-Hansen, 2008. Radiation therapy with tositumomab (B1) anti- cell-cell adhesion in Ewing’s sarcoma cells: actin M., Nylandsted, J., and Jäättelä, M. 2007. Vincristine CD20 monoclonal antibody initiates extracellular and zyxin as key intracellular mediators. Oncogene. induces dramatic lysosomal changes and sensitizes signal-regulated kinase/mitogen-activated 23:5664–5674. cancer cells to lysosome destabilizing siramesine. kinase-dependent cell death that overcomes resis- 19. Fehrenbacher, N., et al. 2008. Sensitization to the Cancer Res. 67:2217–2225.

EBAG9 tempers lymphocyte killing activity Gaël Ménasché1,2 and Geneviève de Saint Basile1,2

1INSERM U768, Paris, France. 2Faculté de Médecine, Université Paris Descartes, Paris, France.

The cytotoxic activity of lymphocytes is crucial for immune surveillance presenting targets. The failure to clear and homeostasis. Several independent, naturally occurring genetic models antigen causes unremitting polyclonal characterized by defects in granule trafficking or exocytosis have helped to CD8+ T cell expansion, activation, and decipher the multiple steps and molecules that regulate the cytotoxic pro- infiltration of visceral organs associated cess. The study by Rüder and colleagues in this issue of the JCI shows that with macrophage activation, with the del- an engineered absence of EBAG9, previously reported as a tumor-associ- eterious release of multiple inflammatory ated antigen, enhances cytotoxic activity of CTLs but not NK cells, likely cytokines including IFN-g (3, 4). acting on the endosomal-lysosomal trafficking of the cytotoxic effectors In addition, the studies of these natural (see the related article beginning on page 2184). This finding adds a new mutants both in humans and in mice have piece to the puzzle of complex mechanisms that tightly regulate the capacity contributed to the characterization of criti- of the cytotoxic response and suggests a new target to negatively modulate cal effectors of the cytotoxic machinery and CTL responsiveness. their function. They also provided evidence of the exquisite regulation of this process, CTLs and NK cells (collectively known as toxic cells have recognized their targets and which occurs in successive steps follow- “cytotoxic lymphocytes”) are major players formed a conjugate, the trafficking of gran- ing cell activation. For instance, studies in the body’s defense against viral infection ule components, including perforin and of Griscelli syndrome type 2, a rare condi- and cancer via their ability to seek out and granzymes, to the immunological synapse tion characterized by partial albinism and kill infected or tumorigenic cells. Although (IS) between the cell and its target, leads occurrence of an HLH syndrome (5), and CTLs are activated by specific antigen rec- to the delivery of perforin and granzymes familial HLH type 3 (FHL3) (6) have shown ognition, the cytotoxic activity of NK cells into the target cells and subsequent target the critical role of two proteins, the small is initiated by specific activating receptors destruction through apoptosis (1, 2). GTPase Rab27a and the priming factor or combinations thereof and is inhibited Studies of natural or engineered Munc13-4, at a late step of the secretory by self MHC class I recognition. Once cyto- mutants involving cytotoxic function tell pathway. Rab27a regulates the tethering of us a great deal about the in vivo function mature cytotoxic granules that have polar- of this pathway. So far, only genetic dis- ized at the IS, whereas Munc13-4 primes Conflict of interest: The authors have declared that no conflict of interest exists. eases leading to the loss of cytotoxic func- the docked cytotoxic granules before Nonstandard abbreviations used: AP1, adaptor tion have been reported (3). These studies their fusion with the plasma membrane protein 1; BLOC-1, biogenesis of lysosome-related have revealed the tremendous importance at the IS. Other proteins, for which their organelles complex 1; EBAG9, receptor–bind- of this cytotoxic pathway in immune defect impairs lymphocyte cytotoxic activ- ing fragment–associated antigen 9; ESCRT, endosomal sorting complex required for transport; HLH, hemo- homeostasis. Congenital defects that lead ity, likely regulate an upstream step in the phagocytic lymphohistiocytosis; HPS2, Hermansky- to either impaired perforin function or cytotoxic pathway. They include adapter Pudlak syndrome type 2; IS, immunological synapse; its dysregulated release result in the spe- protein 3 (AP3), which is deficient in Her- LAMP, lysosomal-associated membrane protein; LCMV, cific severe condition hemophagocytic mansky-Pudlak syndrome type 2 (HPS2) lymphocytic choriomeningitis virus; LRO, lysosome- related organelle; LYST, lysosomal trafficking regulator. lymphohistiocytosis (HLH) syndrome. (7, 8), and the lysosomal trafficking regu- Citation for this article: J. Clin. Invest. 119:2136–2140 This syndrome stems from the inability of lator (LYST) protein, which is deficient in (2009). doi:10.1172/JCI40270. activated cytotoxic cells to clear antigen- Chediak-Higashi syndrome (9, 10). Their

2136 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 8 August 2009 commentaries

Figure 1 Sorting proteins to the cytotoxic granules/secretory lysosomes. The newly synthesized soluble proteins granzymes A and B acquire the man- nose-6-phosphate signal in the Golgi complex, enabling their association with the mannose-6-phosphate receptor (M6PR) in the trans-Golgi network (TGN) and transport to early endosomes before they are sorted to late endosomes. LAMP1 is sorted by AP complexes (not shown) and transported from the plasma membrane to the endosomes, preferentially to the perimeter membrane of the multivesicular bodies (MVBs) in the sorting endosome and then to the late endosome and cytotoxic granule/LRO. Early endosomes sort cargoes to different destinations. Cargoes can be recycled back to the plasma membrane via fast or slow pathways, delivered to the lysosomes through sorting endosomes and multivesicular bodies, or sorted to the trans-Golgi network. BLOC-1 is involved in the exit of cargoes from an early endosome subdomain toward intermediate/sorting endosomes. g2-Adaptin has been reported (19) to associate with the ubiquitin machinery, which directs cargo incorporation into intraluminal vesicles (ILVs) of multivesicular bodies by the ESCRT machinery. The study by Rüder et al. in this issue of the JCI (11) reports that EBAG9 associates with and inhibits g2-adaptin and associates with two BLOC-1 subunits. Loss of EBAG9 enhances the cytotoxic activity of T lymphocytes, likely acting on the endosomal-lysosomal trafficking of the cytotoxic effectors. Although the detailed mechanism accounting for the transport of perforin from the trans-Golgi network to late endosomes is still poorly understood, the findings of Rüder et al. (11) suggest a connection among granzymes and perforin trafficking, the BLOC-1 components, and the ESCRT machinery.

respective defects include missorting of increase in the sorting of cytotoxic effec- cyte function demonstrate a clear immune transmembrane proteins to lysosomes and tor molecules to cytotoxic granules associ- disease, i.e., HLH (12). An “opposite” phe- an increase in the size of cytotoxic granules. ated with EBAG9 deficiency, together with notype, with limitation of the immune Although the precise functions of these the demonstration that EBAG9 interacts response to pathogen, might be seen, as we effectors in the cytotoxic pathway remain with the g2-adaptin adapter protein, indi- discuss below. The trafficking of lysosomal poorly understood, both proteins likely cate that EBAG9 plays a pivotal role in proteins in other cells, such as mast cells, regulate lysosomal biogenesis and mem- the sorting of endosomal-lysosomal pro- may also be increased in EBAG9-deficient brane fusion along the cytotoxic pathway. teins. Therefore, the work presented by mice, potentially exacerbating an anaphy- The proteins mentioned above are all Rüder and colleagues introduces a new lactic reaction. required for proper cytotoxic activity of player involved in the tuning of the cyto- lymphocytes. In contrast, Rüder and col- toxic function of lymphocytes. However, it Biogenesis of cytotoxic granules leagues report in this issue of the JCI that remains to be determined what the precise Mature cytotoxic granules are lysosome- –binding fragment–asso- molecular mechanism is that underlies the related organelles (LROs) that act as dual- ciated antigen 9 (EBAG9) acts as a nega- observed phenotype; there is no evidence function organelles, carrying out both tive regulator of cytotoxic function (11). of immunopathogenesis in EBAG9-defi- degradative and secretory functions (2, Through the generation of Ebag9-defi- cient mice. In the future, it may be worth 13). Cytotoxic granules exhibit an elec- cient mice, they show that loss of EBAG9 studying the consequences of lymphocytic tron-dense core that contains most of the enhances the release of cytotoxic granules choriomeningitis virus (LCMV) infection cytotoxic proteins, including perforin and in vitro as well as the cytotoxic capacity in EBAG9-deficient mice, as in this set- granzymes, similar to secretory organelles. of CTLs from deficient mice in vivo. The ting mice impaired in cytotoxic lympho- The core is surrounded by a multivesicu-

The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 8 August 2009 2137 commentaries lar/lamellar region typical of lysosomes. Which step of the cytotoxic pathway ity and can recruit the ubiquitination The cytotoxic granules have a low lumenal is regulated by EBAG9? machinery (19). Thus, by interacting with pH and contain lysosomal acid hydrolases Rüder and colleagues used various g2-adaptin, EBAG9 may participate in such as cathepsin D and receptors such approaches to determine which step of the endosomal/multivesicular body sort- as a member of the lysosomal-associated cytotoxic granule biogenesis is regulated ing and trafficking system within the membrane protein (LAMP) family. The by EBAG9 expression (11). Their findings ESCRT machinery. This function again biogenesis of these cytotoxic organelles is provide compelling evidence that EBAG9 localizes EBAG9 at the exit of cargo from not well characterized, but, as in the case of does not directly regulate the secretion the early endosome. Of note, depletion of other LROs, they likely use the cooperation of secretory lysosomes, but rather acts g2-adaptin results in an enlargement of of ubiquitous trafficking machineries with upstream of the exocytic process, in the endosomal vesicles and the accumulation cell type–specific cargoes (14) (Figure 1). trafficking of the cytotoxic components in these vesicles of proteins destined to Granzymes, similar to other soluble lyso- along the endosomal-lysosomal pathway. lysosomal degradation (19). In contrast loss somal proteins, are modified during bio- They observed that the loss of EBAG9 of EBAG9 enhances proteolytic processing synthesis by the addition of a mannose- enhanced the sorting of granzyme B and of of the lysosomal protein cathepsin D and 6-phosphate moiety, which is recognized the acid hydrolase cathepsin D to the secre- decreases cytotoxic granule size. This oppo- by mannose-6-phosphate receptors. These tory lysosomes. In contrast, the sorting site phenotype supports a role of EBAG9 as transmembrane receptors cycle among of lysosomal membrane marker LAMP1, a negative regulator of g2-adaptin. the trans-Golgi network and early and late which uses an AP-dependent pathway, was Although our present knowledge of the endosomes, carrying soluble proteins to the reduced in EBAG9-deficient cells. Interest- molecular mechanisms that finely regulate lysosome structures. However, the pathway ingly, they report (although this data is not the intracellular trafficking of proteins in used by perforin to reach the lysosomes is shown) that the sorting of perforin was CTLs precludes further assumption on the still unknown (Figure 1). LAMPs do not also enhanced, suggesting that the sorting precise role of EBAG9 in these cells, some require sorting receptors, since they have a of perforin may occur via the same traffick- points should be underscored. First, the tyrosine-based motif that directly interacts ing pathway as the sorting of granzymes. interaction of EBAG9 with two BLOC-1 with multimeric cytosolic AP1, AP2, and A role for EBAG9 in the trafficking components and with g2-adaptin, if func- AP3. Biogenesis of LROs in different cell between endosomal and lysosomal com- tionally relevant, indicate that EBAG9 types also depends on the function of the partments is further supported by the regulates sorting of proteins from early biogenesis of lysosome-related organelles finding that EBAG9 interacts with differ- endosomes, potentially through two differ- complex-1 (BLOC-1), -2, and -3 protein ent proteins involved in the sorting of pro- ent sorting pathways. Second, it is intrigu- complexes (15). These protein complexes teins at an early step along this route (11). ing to note that the association of an have been largely defined from the studies By yeast two-hybrid analysis, Rüder et al. increase in cytotoxic component traffick- of the various leading to the pheno- identified EBAG9 as interacting with two ing, an enhanced cytotoxic function, and type of HPS or a similar disorder in mice subunits of BLOC-1, snapin and BLOS2, a reduced size of cytotoxic granules, which (14). Of note, the AP3 complex was shown although the authors failed to confirm all result from loss of function of EBAG9, to interact with BLOC-1 on early endo- this interaction by coimmunoprecipita- inversely mirror the phenotypes evoked by somes to facilitate the trafficking of LAMP tion of the proteins expressed at the endog- functional loss of LYST in Chediak-Higashi to the lysosome (16). In addition, cytotoxic enous level (11). BLOC-1 mainly localizes syndrome and of AP3 in HPS2 (8, 20, 21). cells, similar to all cell types, may use the to a subdomain of early endosomes and Both of these proteins are known to partic- endosomal sorting complex required for is involved in the exit of selective cargoes ipate in the sorting of lysosomal proteins transport (ESCRT) (17). Ubiquitylation tar- from this domain to maturing LROs (15). (9, 15). LYST was also recently reported to gets proteins to this pathway. ESCRT com- Although loss of BLOC-1 components has interact with HRS and LIP5, two ESCRT- plexes recognize ubiquitylated proteins on never been shown before to be required associated proteins, and aberrant enlarged early endosomes, which are subsequently for cytotoxic function (18), the possi- vacuolar structures are found when HRS is invaginated into newly forming multi- bility that EBAG9 regulates a BLOC-1– deficient (17, 22). These observations sug- vesicular bodies. Ubiquitylated proteins dependent cytotoxic granule matura- gest opposite functions of EBAG9 (nega- accumulate in these intralumenal vesicles tion, as suggested by these data, remains tive) and LYST and AP3 (positive) in the of endosomes and then are sorted to late an attractive hypothesis. Another role regulatory sorting of endosomal proteins endosomes and lysosomes. So far, none of of snapin in membrane fusion, through into lysosomes. the CTL-restricted lysosomal proteins have its interaction with the SNARE proteins Together with previous observations that been found to follow this ESCRT sorting (SNAP-25 and -23), has also been reported AP3 is able to interact with BLOC-1 and pathway to be incorporated into the vesicu- (15). This interaction may also participate that both HRS and snapin were shown to lar structure of the granules. in cargo exit or travel by regulating mem- interact with SNAP-25 in neuroendocrine Thus different machineries may partici- brane fusion events. cells (15, 22), the work of Rüder et al. (11) pate along the endosomal pathway in the In addition, Rüder et al. show that EBAG9 further suggests that the formation of sorting of cargoes involved in LRO bio- not only associates with BLOC-1 compo- LROs, among them cytotoxic granules, is genesis. The new data reported by Rüder nents but also interacts with the adapter more complicated than that of modified et al. (11) provide some evidence of a link protein g2-adaptin (11). g2-Adaptin is a unit lysosomes. Their work further suggests among EBAG9, the BLOC-1 components, of the AP complexes, but unlike the other that these granules mature by the addi- the ESCRT machinery, and the biogenesis members of the adapter protein family, tion of cargoes sorted through different of cytotoxic granules (Figure 1). g2-adaptin has a ubiquitin-binding abil- pathways and that molecular connections

2138 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 8 August 2009 commentaries between distinct sorting machineries may Rüder and colleagues report that EBAG9- homeostasis. Nat. Rev. Immunol. 6:940–952. 2. Stinchcombe, J.C., and Griffiths, G.M. 2007. Secre- potentially exist. deficient mice exhibited augmented tory mechanisms in cell-mediated cytotoxicity. Together with the previous work of Rüder CTL clearance of antigen when immu- Annu. Rev. Cell Dev. Biol. 23:495–517. and colleagues, which shows that over- nized with Listeria monocytogenes (11). It 3. Menasche, G., Feldmann, J., Fischer, A., and de expression of EBAG9 in neuroendocrine may be worth studying this mouse model Saint Basile, G. 2005. Primary hemophagocytic syndromes point to a direct link between lympho- + cells leads to a decrease in high K –induced to determine whether, during LCMV infec- cyte cytotoxicity and homeostasis. Immunol. Rev. granule release (23), a role for EBAG9 in the tion, enhanced viral antigen clearance has 203:165–179. negative regulation of the cytotoxic activity any consequences on the magnitude or the 4. Fischer, A., Latour, S., and de Saint Basile, G. 2007. Genetic defects affecting lymphocyte cytotoxicity. of lymphocytes is therefore most likely. quality of the immune response. One may Curr. Opin. Immunol. 19:348–353. expect that, in this setting, a rapid elimi- 5. Ménasché, G., et al. 2000. Mutations in RAB27A Does EBAG9 deficiency affect nation of antigens would diminish the cause Griscelli syndrome associated with hemo- phagocytic syndrome. Nat. Genet. 25:173–176. serial killing capacity of CTLs? expansion of antigen-specific effector cells 6. Feldmann, J., et al. 2003. Munc13-4 is essential for CTLs are able to successively kill several or the associated Th1 cytokine production cytolytic granules fusion and is mutated in a form target cells in a short time period. The iter- (e.g., IFN-g, TNF-a, IL-6) and also ulti- of familial hemophagocytic lymphohistiocytosis ative killing ability of these cells depends mately dampen the generation of memory (FHL3). Cell. 115:461–473. 7. Dell’Angelica, E.C., Shotelersuk, V., Aguilar, R.C., + on the combination of two factors: follow- CD8 T cells and modify the immunodom- Gahl, W.A., and Bonifacino, J.S. 1999. Altered ing target cell recognition, TCR trigger- inance hierarchy of the T cell response (i.e., trafficking of lysosomal proteins in Hermansky- ing leads to the release of only a fraction the specificity of the epitopes present in a Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Mol. Cell. 3:11–21. of the mature cytotoxic granules from given antigen that will preferentially elicit 8. Clark, R.H., et al. 2003. Adaptor protein 3-depen- CTLs, and at the same time cytotoxic pro- the CD8+ T cell response), as previously dent microtubule-mediated movement of lytic teins are synthesized to refill the store of shown in mice deficient for IFN-g (26). granules to the immunological synapse. Nat. Immu- nol. 4:1111–1120. cytotoxic components. Regulation of the Alternatively, the increase in the cytotoxic 9. Faigle, W., et al. 1998. Deficient peptide loading quantum of cytotoxic granules secreted granule release by CTLs may cause toxic and MHC class II endosomal sorting in a human at the IS may occur at a very late stage of bystander effects, leading to destruction genetic immunodeficiency disease: the chediak- cytotoxic granule maturation, when these of neighboring cells or tissue infiltrated by higashi syndrome. J. Cell Biol. 141:1121–1134. 10. Ward, D.M., Griffiths, G.M., Stinchcombe, J.C., granules are polarized together with endo- the effector cytotoxic cells. and Kaplan, J. 2000. Analysis of the lysosomal somal exocytic vesicles at the IS and fuse To our knowledge, this report (11) storage disease Chediak-Higashi syndrome. Traf- with these vesicles prior to secretion (24). describes the second non-receptor protein fic. 1:816–822. 11. Rüder, C., et al. 2009. The tumor-associated Thus, a pool of cytotoxic granules ready whose silencing potentiates T lymphocyte antigen EBAG9 negatively regulates the cyto- to release their contents persists in CTLs cytotoxicity. Janus kinase and microtu- lytic capacity of mouse CD8+ T cells. J. Clin. Invest. following killing of a first target. This bule–interacting protein 1 was previously 119:2184–2203. 12. Jordan, M.B., Hildeman, D., Kappler, J., and Mar- pool can then be used to successively kill described as able to restrain T cell–medi- rack, P. 2004. An animal model of hemophago- other encountered targets. An interesting ated cytotoxicity, potentially acting on cytic lymphohistiocytosis (HLH): CD8+ T cells and question is whether the rate of protein cytotoxic granule transport along micro- interferon gamma are essential for the disorder. synthesis is sufficient to compensate for tubules (27). Thus, EBAG9 appears to Blood. 104:735–743. 13. Bossi, G., and Griffiths, G.M. 2005. CTL secretory the increase in trafficking and therefore be a new player able to downregulate the lysosomes: biogenesis and secretion of a harmful release of cytolytic components when process of lymphocyte cytotoxic activity. It organelle. Semin. Immunol. 17:87–94. EBAG9 is defective. suggests that CTL activity, as other major 14. Raposo, G., Marks, M.S., and Cutler, D.F. 2007. Lysosome-related organelles: driving post-Golgi effector pathways of the immune response, compartments into specialisation. Curr. Opin. Cell Could excessive cytotoxic function is controlled at multiple checkpoints. It is Biol. 19:394–401. of lymphocytes disturb not foolish to predict a future description 15. Setty, S.R., et al. 2007. BLOC-1 is required for cargo- specific sorting from vacuolar early endosomes immune homeostasis? of several of these checkpoints. toward lysosome-related organelles. Mol. Biol. Cell. The granule-dependent cytotoxic activity 18:768–780. of lymphocytes plays a major role in the Acknowledgments 16. Di Pietro, S.M., et al. 2006. BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate elimination of infected cells, simultaneous- We thank Alain Fischer for critical reading protein trafficking on endosomes. Mol. Biol. Cell. ly regulating the magnitude of the immune of this manuscript. The authors are sup- 17:4027–4038. response. LCMV infection in mice defi- ported by grants from the French National 17. Williams, R.L., and Urbe, S. 2007. The emerging cient for the cytotoxic activity of lympho- Institute for Health and Medical Research shape of the ESCRT machinery. Nat. Rev. Mol. Cell Biol. 8:355–368. cytes reproduces the immunopathology of (INSERM), the French National Research 18. Bossi, G., et al. 2005. Normal lytic granule secre- HLH observed in humans (12, 25). Not all Agency (ANR), and the Fondation pour la tion by cytotoxic T lymphocytes deficient in strains of viruses can trigger such immune Recherche Médicale (FRM). BLOC-1, -2 and -3 and myosins Va, VIIa and XV. Traffic. 6:243–251. manifestations in mice deficient for cyto- 19. Rost, M., Doring, T., and Prange, R. 2008. gamma2- toxic lymphocyte activity. The fact that Address correspondence to: Geneviève de Adaptin, a ubiquitin-interacting adaptor, is a sub- clearance of LCMV infection is absolutely Saint Basile, INSERM U768, 149 rue de strate to coupled ubiquitination by the ubiquitin ligase Nedd4 and functions in the endosomal path- dependent on perforin-mediated cytolysis Sèvres, 75015, Paris, France. Phone: 33-1- way. J. Biol. Chem. 283:32119–32130. may be an important parameter. Thus, in 44-49-50-80; Fax: 33-1-42-73-06-40; E-mail: 20. Barbosa, M.D.F.S., et al. 1996. Identification of the the context of LCMV infection, the balance [email protected]. homologous beige and Chediak-Higashi syndrome between cytotoxicity, antigen presenta- genes. Nature. 382:262–265. 21. Nagle, D.L., et al. 1996. Identification and muta- tion, and cytokine production determines 1. Voskoboinik, I., Smyth, M.J., and Trapani, J.A. 2006. tion analysis of the complete for Chediak- the outcome of the immune response. Perforin-mediated target-cell death and immune Higashi syndrome. Nat. Genet. 14:307–311.

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22. Tchernev, V.T., et al. 2002. The Chediak-Higashi ule maturation and exocytosis require the effector 2000. Regulation of antigen-specific CD8+ T cell protein interacts with SNARE complex and signal protein hMunc13-4. Nat. Immunol. 8:257–267. homeostasis by perforin and interferon-gamma. transduction proteins. Mol. Med. 8:56–64. 25. Pachlopnik Schmid, J., et al. 2008. A Griscelli Science. 290:1354–1358. 23. Ruder, C., et al. 2005. EBAG9 adds a new layer of con- syndrome type 2 murine model of hemophago- 27. Libri, V., et al. 2008. Jakmip1 is expressed upon trol on large dense-core vesicle exocytosis via interac- cytic lymphohistiocytosis (HLH). Eur. J. Immunol. T cell differentiation and has an inhibitory func- tion with Snapin. Mol. Biol. Cell. 16:1245–1257. 38:3219–3225. tion in cytotoxic T lymphocytes. J. Immunol. 24. Menager, M.M., et al. 2007. Secretory cytotoxic gran- 26. Badovinac, V.P., Tvinnereim, A.R., and Harty, J.T. 181:5847–5856.

Haptoglobin halts hemoglobin’s havoc Gregory J. Kato

Sickle Cell Vascular Disease Section, Pulmonary and Vascular Medicine Branch, National Heart, Lung, and Blood Institute, and Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.

Hemoglobin (Hb) is crucial to the function of the red blood cell. However, Sickle-cell disease has provided the most when it is released during intravascular hemolysis from the cell into blood well-documented example of chronic hemo- plasma, it produces a state of NO depletion, oxidant stress, and vascular dys- lysis and clinical vasculopathy, with markers function, including hypertension. In their study reported in this issue of the of hemolysis-induced NO scavenging statis- JCI, Boretti and colleagues used canine and guinea pig models to demon- tically linked with pulmonary hypertension, strate that pharmacological doses of glucocorticoid can increase the plasma leg ulceration, priapism, and cerebrovascu- levels of haptoglobin (Hp), the principal plasma-binding protein for free Hb lar disease (5). Reports also have begun to (see the related article beginning on page 2271). Hp prevented Hb-induced accumulate for these same exact complica- hypertension and the generation of oxidant damage to the kidney. Neutral- tions, arising in other non-sickling, acute, ization of free Hb appears to be part of the downstream antiinflammatory and chronic hemolytic disorders, support- properties of glucocorticoid. ing a contributory role for hemolysis in the pathophysiology of this vasculopathy syn- Hemoglobin (Hb) plays a critical role in Hemolysis, NO scavenging, oxidative drome (5). This growing list includes thal- vascular function — it carries oxygen and stress, and clinical outcomes assemia, autoimmune hemolytic anemia, mediates adaptive vasodilation via NO For decades, it has been known that Hb malaria, paroxysmal nocturnal hemoglo- signaling, most likely via nitrite reduc- is a highly efficient scavenger of NO in binuria, unstable hemoglobinopathy, and tase activity, under hypoxic conditions vitro. However, only in recent years has it hereditary membranopathies (5). (1). When red blood cells lyse during become more widely appreciated that cell- The biological importance of these path- intravascular hemolysis, Hb crosses the free Hb scavenges NO in vivo in human ways is emphasized by the redundant and red blood cell membrane into plasma. disease and produces clinical manifesta- overlapping mechanisms that have evolved Once decompartmentalized from red tions (Figure 1) (2). The depletion of NO, to detoxify cell-free plasma Hb. First among blood cells, free Hb potently induces oxi- a crucial endogenous antioxidant, is com- these is Hp, a tetrameric plasma glycopro- dative stress and scavenges NO, reducing pounded by the oxidative stress induced by tein that binds cell-free plasma Hb and the bioavailability of this critical antiox- decomposition of Hb into heme and ele- quickly escorts it to the CD163 protein, in idant and master regulator of vascular mental iron, highly oxidative co-conspira- which the Hp-Hb complex is avidly bound homeostasis, wreaking havoc on vascular tors against vascular health. Indeed, not and cleared from plasma, depleting plasma health (2). In this issue of the JCI, Boret- only does a canine model of intravascular Hp in the process (6) (Figure 1). Boretti ti et al. show, in canine and guinea pig hemolysis acutely demonstrate the induc- et al. (3) found that Hp binding to Hb is models, that pharmacological activation tion of systemic and pulmonary hyperten- sufficient to prevent the generation of oxi- of the glucocorticoid pathway induces sion but also impaired creatinine clear- dant species from cell-free Hb that would expression of the Hb-scavenging protein ance, presumably due to decreased renal otherwise mediate hypertension and other haptoglobin (Hp), which sequesters cell- blood flow (4). Overlapping syndromes adverse vascular outcomes, perhaps in part free plasma Hb and neutralizes much of have been observed in other human exam- by sequestering Hb in a high-molecular- its vasculotoxic oxidative stress, protect- ples of acute hemoglobinemia (excess Hb weight complex that would not extravasate ing against systemic hypertension and in the blood plasma), such as the acute into the subendothelial space. Interestingly, other vasculopathic outcomes (3). hemolytic transfusion reaction (abrupt Boretti et al. also show that Hp-bound hemolysis of transfused red blood cell), or Hb has a very high oxygen affinity, which Conflict of interest: The author has received research following infusion of the first generation should correspond to increased nitrite support in the form of a Collaborative Research and of cell-free blood substitutes (2). The acute reductase activity (1), potentially stimulat- Development Agreement between the National Insti- experimental canine vasculopathy is par- ing the activity of Hp-Hb complexes to pro- tutes of Health and Ikaria–INO Therapeutics. tially reversed by administration of NO (4) duce the endogenous antioxidant NO from Nonstandard abbreviations used: Hb, hemoglobin; Hp, haptoglobin. or alternatively, as described in this issue nitrite. As part of the teleological evidence Citation for this article: J. Clin. Invest. 119:2140–2142 by Boretti et al., by glucocorticoid-induced of the biological influence of cell-free plas- (2009). doi:10.1172/JCI40258. production of endogenous Hp (3). ma Hb, additional Hb-binding pathways

2140 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 8 August 2009