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Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008;36:296-327. 6. Beale RJ, Hollenberg SM, Vincent JL, Parrillo JE. Vasopressor and inotropic support in septic shock: an evidence-based review. Crit Care Med 2004;32:Suppl:S455-S465. 7. Rivers E, Nguyen B, Havstad S, et al. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001;345:1368-77. 8. Trzeciak S, Dellinger RP, Abate NL, et al. Translating research to clinical practice: a 1-year experience with implementing early goal-directed therapy for septic shock in the emergency department. Chest 2006;129:225-32. 9. Kumar A, Roberts D, Wood KE, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;34:1589-96. 10. Parrillo JE. Approach to the patient with shock. In: Goldman

L, Ausiello D, eds. Cecil texbook of medicine. 23rd ed. Vol. 1. Philadelphia: Saunders-Elsevier, 2008:742-50. 11. Hayes MA, Timmins AC, Yau EHS, Palazzo M, Hinds CJ, Watson D. Elevation of systemic oxygen delivery in the treatment of critically ill patients. N Engl J Med 1994;330:1717-22. 12. Landry DW, Oliver JA. The pathogenesis of vasodilatory shock. N Engl J Med 2001;345:588-95. 13. Dnser MW, Mayr AJ, Ulmer H, et al. Arginine vasopressin in advanced vasodilatory shock: a prospective, randomized, controlled study. Circulation 2003;107:2313-9. 14. Malay MB, Ashton RC Jr, Landry DW, Townsend RN. Lowdose vasopressin in the treatment of vasodilatory septic shock. J Trauma 1999;47:699-705. 15. Russell JA, Walley KR, Singer J, et al. Vasopressin versus norepinephrine infusion in patients with septic shock. N Engl J Med 2008;358:877-87.
Copyright 2008 Massachusetts Medical Society.

Collaboration, Genetic Associations, and Lupus Erythematosus

Mary K. Crow, M.D. Systemic lupus erythematosus (SLE), a disease that preferentially targets women during the reproductive years, is considered by many clinicians and investigators to be the prototypic autoimmune disease. Among clinicians, this status is based on the characteristic involvement of multiple organ systems most notably, skin, kidneys, joints, central nervous system, and cardiovascular system with the deposition of immune complexes and complement, inflammation, and vascular damage noted by pathologists. From the perspective of the immunologist, SLE is a model disease that has provided important insights into immune-system function. As is characteristic of most complex diseases, genetic and environmental factors determine the development of SLE and what its clinical manifestations will be. Recent technological advances have allowed rapid and increasingly cost-efficient analysis of single-nucleotide polymorphisms (SNPs) in patients with complex diseases and appropriate control subjects. This week, important new data from two complementary genomewide association studies of patients with SLE,1,2 from a third genomewide study that focused on nonsynonymous DNA variations,3 and an analysis of an attractive candidate gene4 are published in the Journal and in Nature Genetics. Results from these ambitious projects involving international collaborations expand a growing compendium of genetic data that implicate many components of the immune system in the pathogenesis of SLE (Table 1). Recognition of the essential role of innate
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immune-system activation in SLE and other immune-mediated diseases has followed the characterization of toll-like receptors and their environmental and endogenous stimuli. Production of type I interferon in patients with SLE is now recognized as a central pathogenic mechanism,5 and increased serum interferon activity is a heritable trait in families with a history of lupus (Fig. 1).6 Analysis of genes encoding components of the interferon pathway has led to extensive support for an association of polymorphic variants of interferon regulatory factor 5 (IRF5) with SLE.7 The IRF5 association is replicated in both genomewide association studies reported this week,1,2 although a functional link between the IRF5 risk haplotype and increased production of type I interferon has yet to be made. The central contribution of the adaptive immune response to SLE is represented by characteristic autoantibodies specific for nucleic-acid containing particles (Fig. 1). The HLA locus that generates the strongest statistical association with SLE has been associated with the production of particular autoantibodies,8 suggesting that MHC class II molecules promote the expansion of autoantigen-specific T cells and the production of T-celldependent autoantibodies. Moreover, variations in other lupus-associated genes encode proteins expressed in T and B cells that are associated with altered activation or function of those cells. Protein tyrosine phosphatase, nonreceptor type 22 (PTPN22), for example, encodes a cytoplasmic lymphoid phosphatase expressed

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Table 1. New and Confirmed Genetic Variants Conferring a Significant Risk of Systemic Lupus Erythematosus in Two Genomewide Association Studies.* Gene HLA HLA ITGAM IRF5 KIAA1542 PXK PTPN22 FCGR2A STAT4 BLK Genome Location 6p21.33 6p21.32 16p11.2 7q32.1 11p15.5 3p14.3 1p13 1q23 2q32 8p23.1 Presentation of antigen Presentation of antigen Adhesion of leukocytes to endothelial cells Production of interferon- Linkage disequilibrium with IRF7; production of type I interferon Unknown effect of serinethreonine kinase Inhibition of lymphocyte activation Clearance of immune complexes Modulation of the production of cytokines in T cells and natural killer cells; activation of response of macrophages to interferon- Activation of B cells Proposed Function

* Data are from Hom et al.1 and from the International Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN) study.2 This variant meets the authors criteria for association with systemic lupus erythematosus only in the SLEGEN study. This variant meets the authors criteria in both Hom et al. and the SLEGEN study. This variant meets the authors criteria only in Hom et al., although the SLEGEN study provides suggestive evidence of an association at this locus.

in hematopoietic cells, mediates interaction with Csk tyrosine kinase, and inhibits antigen-induced activation of T cells. Perhaps surprisingly, the lupus-associated allele of PTPN22 encodes a more active phosphatase and is associated with decreased lymphocyte activation.9,10 Signal transducer and activator of transcription 4 (STAT4) encodes an important signaling molecule that regulates immune function, possibly through its effect on cytokine production by T cells and natural killer cells.11 Both of the new genomewide association studies confirm associations of HLA and STAT4 variants with SLE (although the study that originally implicated STAT4 in lupus11 and one of the newly published genomewide studies1 share some of the same patients), and the study by the International Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN)2 also confirms the role of PTPN22. The association between lupus and the B-cell specific tyrosine kinase gene (BLK ), described in this issue of the Journal by Hom et al.,1 is novel. BLK protein is involved in the adaptive immune response and is a member of the Src kinase family (along with Fyn, Lck, and Lyn). Ligation of the B-cell surface immunoglobulin receptor (BCR) triggers an interaction between BLK and the Ig- and Ig- components of the BCR complex, phosphorylation of BLK, and activation of its kinase

function.12 BLK associates with the tyrosine kinase Syk and can phosphorylate inhibitory Fc receptors on B cells, indicating that BLK may mediate both positive and negative regulatory effects. Expression of an active BLK protein in the mouse promotes B-cell maturation that mimics responses typically generated by pre-BCR,13 suggesting that altered BLK expression or function in SLE might affect the development of the B-cell repertoire as well as mature B-cell function. Gene-expression studies performed by Hom et al. on cell lines transformed by EpsteinBarr virus and bearing the risk allele showed decreased BLK expression, but investigation of primary cells from patients with SLE and control subjects will be required to develop hypotheses about function on the basis of the genetic data. Additional new information from a genomewide association study of nonsynonymous SNPs in Swedish patients with SLE implicates another B-cell molecule, the B-cell scaffold protein with ankyrin repeats 1 (BANK1).3 BANK1 is an adapter protein that links the activation of Src family tyrosine kinases by B-cellreceptor ligation to calcium-channel mobilization.14 Mice that are deficient in BANK1 have shown spontaneous formation of germinal centers and augmented proliferative responses to CD40 ligation.15 The new data associating BLK and BANK1 variants with
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SLE draw renewed attention to the molecular pathways that mediate B-cell responses to antigen and T-cell help. The effector mechanisms that are stimulated by antibodies and cytokines produced by a dysregulated adaptive immune response contribute to inflammation and tissue damage in patients with SLE. Deficiencies of complement components that have an effect on the solubility and clearance of immune complexes and cellular debris are well-documented genetic contributors to lupus. Polymorphisms in several of the Fc receptors for immunoglobulin have been associated with SLE,16 and the genomewide study by SLEGEN confirms a significant association between the Fc receptor for IgG (FCGR2A) and SLE. Perhaps the strongest association uncovered by the new genetic analyses is that between disease and ITGAM, the gene encoding integrin alpha M (also known as CD11b, Mac-1, and complement receptor type 3). Additional ligands include fibrinogen, platelet glycoprotein 1b, lipoprotein(a), and intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2).17,18 Although the specific SNPs in ITGAM that are most significantly associated with SLE in three of the new studies1,2,4 are distinct, the authors of each study conclude that genetic variants could confer changes in amino acids that have an effect on binding, function, or both. Two of the studies (by SLEGEN and by Nath et al.4) provide the strongest support for an association between a nonsynonymous SNP in ITGAM and SLE. These two studies shared one third of 9073 samples from case subjects and control subjects that were studied, although a combined analysis of the 7380 independent samples generated a maximum combined P=2.021026 and an odds ratio of 1.65 for the association between the T allele of rs9888739 and lupus.2 Nath et al. then designated rs1143679 as a candidate causal SNP in ITGAM in patients with SLE who are of European or African descent. This variant encodes an amino-acid change from arginine to histidine at position 77, an alteration that may modify the conformation of the proteins 1 domain, the region responsible for binding ICAM-1. (Complement C3 fragment iC3b binds to a distinct site.) In view of the numerous ligands that can pair with this integrin, several hypotheses can be enter-

Figure 1 (facing page). A Model of the Pathogenesis of Systemic Lupus Erythematosus (SLE) That Implicates the Products of Disease-Associated Polymorphic Genes. Candidate environmental triggers of SLE include ultraviolet light, demethylating drugs, and infectious or endogenous viruses or viral-like elements. These stimuli induce apoptosis or generate stimulatory DNA or RNA that activates the innate immune response through pathways that are either dependent or independent of toll-like receptors, leading to secretion of type I interferon. Pathogenic variants of interferon regulatory factor 5 (IRF5 ) and possibly of IRF7 contribute to augmented production of interferon- by plasma cytoid dendritic cells and an increased level of antigen presentation by myeloid dendritic cells. Impaired function of molecular components of DNA checkpoint pathways, such as 3 repair exonuclease 1 (TREX1), also generates stimulatory DNA that induces production of interferon-. Impaired clearance of apoptotic or necrotic debris, as might occur with rare deficiencies of complement components, can provide sufficient self-antigen for effective presentation to T cells. Allelic products of the HLA locus include MHC class II molecules that preferentially present disease-associated self-antigens, perhaps released from apoptotic cells, to self-reactive T cells. Activation of T cells in the course of an adaptive immune response to self-antigens can be modified by regulators of intracellular signaling pathways. PTPN22, which encodes a lymphoid phosphatase, associates with CBL, CSK, and GRB2, modulating activation of T and B cells. Altered expression or function of STAT4 (signal transducer and activator of transcription 4) in antigen-presenting cells, T cells, or natural killer (NK) cells modifies the profile of cytokines generated, promoting interferon- production in response to interferon-. T-celldependent activation of B cells and autoantibody production are amplified by altered expression or function of BLK (B-cellspecific tyrosine kinase) and BANK1 (B-cell scaffold protein with ankyrin repeats 1). Once autoantibodies have been generated, immune complexes containing nucleic acids form, stimulate toll-like receptors, and amplify production of interferon-. Pathogenic variants of the Fc receptor for IgG (FCGR2A) may not adequately clear the immune complexes. The complexes accumulate in target tissues, including skin and blood vessels, which incites diffuse vasculopathy. Pathogenic ITGAM variants that alter binding to ICAM-1 (intercellular adhesion molecule 1) increase the adherence of leukocytes to endothelial cells activated by antibodies or cytokines, which promotes vascular disease. Given the prominent vasculopathy associated with mutations in TREX1 in some patients, impaired DNA degradation and chronic DNA checkpoint activation preferentially occur in endothelial cells and focus immune-mediated damage on the vasculature. PMN denotes polymorphonuclear cell.

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tained to explain the association between ITGAM and SLE. Impaired clearance of immune complexes would be consistent with the contribution of excess immune complexes to inflammation and tissue damage in SLE. But if the predictions of Nath et al. regarding the effect of the amino acid 77 polymorphism on the ICAM-1binding

site are supported experimentally, attention is drawn to integrin-mediated interactions between leukocytes and endothelial cells and their role in the vasculopathy and vasculitis of SLE. Autoantibodies can trigger endothelial-cell expression of ICAM-1 and neutrophil expression of M-2 integrin, but antibody-independent mechanisms

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of vascular insult in SLE have been proposed by patients with the most severe disease: those of Buyon et al.19 and Belmont and Abramson20 and African, Hispanic, and Asian descent. involve the same adhesive interactions. Dr. Crow reports having equity interest in XDx, being an inThe identification of ITGAM as a lupus-asso- ventor on an application for a patent on an assay for type I interferon, and serving on an advisory board for the Alliance for ciated gene should rekindle interest in and in- Lupus Research. No other potential conflict of interest relevant vestigation of the mechanisms underlying many to this article was reported. characteristic clinical and pathologic features of article (10.1056/NEJMe0800096) was published at www. SLE including onionskinning of splenic ar- This nejm.org on January 20, 2008. terioles, retinopathy, livedo reticularis, the wireloop lesions of glomerular capillaries, and pre- From the Mary Kirkland Center for Lupus Research, Hospital mature atherosclerosis (the latter being one of for Special Surgery, New York. the most important causes of morbidity and mor1. Hom G, Graham RR, Modrek B, et al. Association of systality associated with lupus). Recent studies21,22 temic lupus erythematosus with C8orf13BLK and ITGAMITGAX. of rare mutations in the 3 repair exonuclease 1 N Engl J Med 2008;358:900-9. (TREX1) gene, encoding a DNase, in association 2. International Consortium for Systemic Lupus Erythematosus Genetics, Harley JB, Alarcon-Riquelme ME, et al. A genomewith lupus and retinal vasculopathy, provide an wide association scan in women with systemic lupus erythemaadditional rationale for the study of mechanisms tosus identifies risk variants in ITGAM, PXK, KIAA1542 and other loci and confirms multiple loci contributing to disease of vascular disease in SLE. susceptibility. Nat Genet (in press). All together, the new data highlight the ex- 3. Kozyrev SV, Abelson A-K, Wojcik J, et al. Functional variants tensive variability in genes encoding mediators of in the B cell gene BANK1 are associated with systemic lupus innate and adaptive immune responses, includ- erythematosus. Nat Genet (in press). 4. Nath SK, Han S, Kim-Howard X, et al. A non-synonymous ing components of the signaling pathways that functional variant in integrin--M (ITGAM) is associated with regulate lymphocyte activation and the cell-surface systemic lupus erythematosus. Nat Genet (in press). receptors that generate tissue responses. The ge- 5. Rnnblom L, Eloranta ML, Alm GV. The type I interferon system in systemic lupus erythematosus. Arthritis Rheum 2006; netic diversity that is described in these and other 54:408-20. recent studies can be viewed as essential for main- 6. Niewold TB, Hua J, Lehman TJ, Harley JB, Crow MK. High taining adequate host defense against infectious serum IFN-alpha activity is a heritable risk factor for systemic lupus erythematosus. Genes Immun 2007;8:492-502. microbes at the level of the aggregate human 7. Sigurdsson S, Gring HH, Kristjansdottir G, et al. Comprepopulation. When the combination of variations hensive evaluation of the genetic variants of interferon regulafavors immune-system activation, inflammation, tory factor 5 reveals a novel 5bp length polymorphism as strong risk factor for systemic lupus erythematosus. Hum Mol Genet and vascular damage, SLE can result. Fruitful (in press). areas for further study are identified by the re- 8. Graham RR, Ortmann W, Rodine P, et al. Specific combinasults of Hom et al. and SLEGEN and include the tions of HLA-DR2 and DR3 class II haplotypes contribute graded risk for disease susceptibility and autoantibodies in human SLE. B-cell-receptorsignaling pathway and the mech- Eur J Hum Genet 2007;15:823-30. anisms of adhesion of inflammatory cells to the 9. Vang T, Congia M, Macis MD, et al. Autoimmune-associated vasculature. The documentation of genetic as- lymphoid tyrosine phosphatase is a gain-of-function variant. Nat Genet 2005;37:1317-9. sociations is only the first step in defining how 10. Rieck M, Arechiga A, Onengut-Gumuscu S, Greenbaum C, the implicated molecular pathways contribute to Concannon P, Buckner JH. Genetic variation in PTPN22 corredisease. Ultimately, the goal should be to iden- sponds to altered function of T and B lymphocytes. J Immunol 2007;179:4704-10. tify therapies based on full knowledge of the 11. Remmers EF, Plenge RM, Lee AT, et al. STAT4 and the risk of molecular pathways relevant to all patients with rheumatoid arthritis and systemic lupus erythematosus. N Engl lupus, regardless of their ancestral origin (three J Med 2007;357:977-86. 12. Aoki Y, Kim YT, Stillwell R, Kim TJ, Pillai S. The SH2 doof the current studies1-3 were restricted to patients mains of Src family kinases associate with Syk. J Biol Chem of European descent). 1995;270:15658-63. The new studies also highlight the value of 13. Tretter T, Ross AE, Dordai DI, Desiderio S. Mimicry of pre-B cell receptor signaling by activation of the tyrosine kinase Blk. structured international collaborations that make J Exp Med 2003;198:1863-73. use of and recognize diverse talents and efforts. 14. Yokoyama K, Su Ih IH, Tezuka T, et al. BANK regulates BCRThese studies, which involved investigators from induced calcium mobilization by promoting tyrosine phophorylation of IP(3) receptor. EMBO J 2002;21:83-92. academic centers in the United States and Europe, 15. Aiba Y, Yamazaki T, Okada T, et al. BANK negatively reguhave been supported by government agencies, lates Akt activation and subsequent B cell responses. Immunity research foundations, and industry. Such collab- 2006;24:259-68. 16. Salmon JE, Millard S, Schacter LA, et al. Fc gamma RIIA orative approaches will be needed for future alleles are heritable risk factors for lupus nephritis in African studies of SLE genetics that are focused on the Americans. J Clin Invest 1996;97:1348-54.
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17. Wang Y, Sakuma M, Chen Z, et al. Leukocyte engagement of

platelet glycoprotein Ibalpha via the intergrin Mac-1 is critical for the biological response to vascular injury. Circulation 2005;112:2993-3000. 18. Sotiriou SN, Orlova VV, Al-Fakhri N, et al. Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin. FASEB J 2006;20:559-61. 19. Buyon JP, Shadick N, Berkman R, et al. Surface expression of Gp 165/95, the complement receptor CR3, as a marker of disease activity in systemic lupus erythematosus. Clin Immunol Immunopathol 1988;46:141-9. 20. Belmont HM, Abramson SB. Mechanisms of acute inflam-

mation and vascular injury in systemic lupus erythematosus. In: Wallace DJ, Hahn BH, eds. Dubois lupus erythematosus. 7th ed. Philadelphia: Lippincott Williams & Wilkins, 2007:23654. 21. Lee-Kirsch MA, Gong M, Chowdhury D, et al. Mutations in the gene encoding the 3-5 DNA exonuclease TREX1 are associated with systemic lupus erythematosus. Nat Genet 2007;39:1065-7. 22. Richards A, van den Maagdenberg AM, Jen JC, et al. C-terminal truncations in human 3-5 DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy. Nat Genet 2007;39:1068-70.
Copyright 2008 Massachusetts Medical Society.

Complexities of Prostate-Cancer Risk

Edward P. Gelmann, M.D. As men age, prostate epithelial cells are subjected to substantial stresses, and these stresses can damage DNA, thereby causing cellular transformation. The aging prostate gland acquires numerous foci of cancer cells that arise from distinct clonal transformation events.1 That most of these foci never develop into clinically detectable cancer is consistent with the frequent finding of prostate cancer during autopsies of asymptomatic men in whom the condition was never diagnosed.2 Susceptibility to prostate cancer has a clear heritable component: men are at an increased risk for the disease if they have a first-degree relative with prostate cancer. The more relatives a man has with the disease, the greater his risk of prostate cancer.3 The hereditary aspect of prostate cancer, particularly in families in which the disease was diagnosed before the age of 60 years, led to genetic linkage studies that identified several candidate tumor-suppressor genes. However, the role of some of these genes in causing cancer was not confirmed by subsequent analyses. One gene that was identified in linkage studies, HPC1, codes for RNase-L protein, a mediator of the action of interferon. RNase-L could affect carcinogenesis in the prostate because of its role in inflammation or by attenuating surveillance of infection by a gammaretrovirus,4-6 but these possibilities are unproven. Many of the other loci implicated by linkage analysis are limited to very few prostate-cancer kindreds. The mapping of the human genome allows for genomewide association studies that reveal genetic determinants of disease on a much larger scale than do traditional linkage studies. Such studies have found a genetic influence in prostate cancer, even in men without a family history. Single-nucleotide polymorphisms (SNPs) at three chromosomal loci 8q24, 17q12, and 17q24.3 have reproducibly scored as loci associated with prostate cancer.7-9 However, the loci of these SNPs do not reside within or near identifiable genes. It has been hypothesized that they exist in regulatory regions of DNA that control gene expression. Such regions may contain enhancers that affect the transcription of remote genes on the same chromosome. Alternatively, they could be in regions of DNA that code for microRNAs or other regulatory transcripts that influence the expression of genes on other chromosomes, the stability of messenger RNA, or even the fate of proteins. The three 8q24 loci are each independently linked to the risk of prostate cancer, and when more than one high-risk allele is present, the risk is magnified in proportion to the number of high-risk loci. These findings have important implications for elucidating the function of the three loci. In this issue of the Journal, the study by Zheng et al.10 extends our knowledge of the genetic predisposition to prostate cancer by examining the association between prostate cancer and five SNPs that map to the three 8q24 loci, to 17q12, and to 17q24.3. The investigators first examined the prostate-cancer risk ratios for 16 polymorphic markers at the three loci to determine which individual SNP was most strongly associated with prostate cancer for each locus. The risk ratios for the 16 SNPs ranged from 1.07 to 1.65. One SNP was chosen as most highly representative of each of the five high-risk loci. Individually, the risk ratios associated with these loci ranged from 1.22 to 1.53. When four or five high-risk genotypes were present, they were associated with a composite
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