Letters to the Editor
Acknowledgements We thank all SLE patients and controls for making this study possible. This work was supported by grant SAF2006-00398 from Plan Nacional de IþDþI and by the Junta de Andalucı´ a, grupo CTS-180. Disclosure statement: The authors have declared no conflicts of interest. E. SA´NCHEZ, J. M. SABIO1, J. JIME´NEZ-ALONSO1, J. L. CALLEJAS2, M. CAMPS3, E. DE RAMO´N3, R. GARCI´A-PORTALES4, M. DE HARO4, N. ORTEGO-CENTENO2, M. A. LO´PEZ-NEVOT5, J. MARTI´N Consejo Superior de Investigaciones Cientı´ficas, CSIC, 1Servicio de Medicina Interna, Hospital Virgen de las Nieves, 2Servicio de Medicina Interna, Hospital Clı´nico San Cecilio, Granada, 3Servicio de Medicina Interna, Hospital Carlos-Haya, Malaga, 4Servicio de Reumatologı´a, Hospital Virgen de la Victoria, Ma´laga and 5Servicio de Inmunologı´a, Hospital Virgen de las Nieves, Granada, Spain Accepted 1 August 2007 Correspondence to: E. Sa´nchez, Instituto de Parasitologı´ a y Biomedicina ‘Lo´pez-Neyra’, CSIC, Parque Tecnolo´gico de Ciencias de la Salud., Avenida del Conocimiento s/n 18100Armilla (Granada), Spain. E-mail:
[email protected] 1 Gaffney PM, Ortmann WA, Selby SA et al. Genome screening in human systemic lupus erythematosus: results from a second Minnesota cohort and combined analyses of 187 sib-pair families. Am J Hum Genet 2000;66:547–56. 2 Reith W, Mach B. The bare lymphocyte syndrome and the regulation of MHC expression. Annu Rev Immunol 2001;19:331–73. 3 Swanberg M, Lidman O, Padyukov L et al. MHC2TA is associated with differential MHC molecule expression and susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction. Nat Genet 2005;37:486–94. 4 Akkad DA, Jagiello P, Szyld P et al. Promoter polymorphism rs3087456 in the MHC class II transactivator gene is not associated with susceptibility for selected autoimmune diseases in German patient groups. Int J Immunogenet 2006;33:59–61. 5 Orozco G, Robledo G, Linga Reddy MV et al. Study of the role of a functional polymorphism of MHC2TA in rheumatoid arthritis in three ethnically different populations. Rheumatology 2006;45:1442–4. 6 Martinez A, Sanchez-Lopez M, Varade J et al. Role of the MHC2TA gene in autoimmune diseases. Ann Rheum Dis 2007;66:325–9. 7 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725. 8 Sanchez E, Gomez LM, Lopez-Nevot MA et al. Evidence of association of macrophage migration inhibitory factor gene polymorphisms with systemic lupus erythematosus. Genes Immun 2006;7:433–6. 9 Eyre S, Bowes J, Spreckley K et al. Investigation of the MHC2TA gene, associated with rheumatoid arthritis in a Swedish population, in a UK rheumatoid arthritis cohort. Arthritis Rheum 2006;54:3417–22. 10 Koizumi K, Okamoto H, Iikuni N et al. Single nucleotide polymorphisms in the gene encoding the major histocompatibility complex class II transactivator (CIITA) in systemic lupus erythematosus. Ann Rheum Dis 2005;64:947–50.
Rheumatology 2008;47:103–104 doi:10.1093/rheumatology/kem259 Advance Access publication 22 November 2007
The candidate lupus susceptibility gene Ifi202a is largely dispensable for B-cell function SIR, The transcriptional modulator Ifi202 has been implicated in the pathogenesis of lupus via genetic studies in the vicinity of the Nba2 locus [1]. It is a member of the interferon-inducible Ifi200 cluster of genes located on mouse chromosome 1, which bears homology to a cluster of three human chromosome 1 genes (MNDA, IFI 16 and AIM2 [2]), all of which share a largely conserved 200 amino acid domain and have been implicated in multiple cellular processes, including cellular proliferation and apoptosis, by virtue of their ability to modulate the activity of relevant transcriptional regulators such as p53 [3], Rb, E2F [4] and NF-B [5]. In lupus, Ifi202 has been proposed to regulate apoptosis in B-cells, perhaps via modulation of p53 [1, 6], thereby contributing to the underlying B-cell hyperactivity that causes disease. In addition, Ifi202 appears to be up-regulated in response
103
to IL-6, which has also been implicated in lupus pathogenesis [7], and may be a target of the lupus autoantibody response, at least in mice [8]. However, much of the evidence linking Ifi202 and lupus pathogenesis has remained largely correlative and is based upon studies with Nba2 congenic mice, where a non-coding singlenucleotide polymorphism (SNP) appears to modulate the expression of Ifi202 [1, 6]. To explore the role of Ifi202 in immunity, we examined the B-cell phenotype of Ifi202a-deficient (Ifi202KO) mice [9], expecting to observe attenuated B-cell activation, correlating with the findings in Nba2 animals, which exhibit elevated Ifi202 expression [1]. This study was approved by and performed according to International Animal Care and Use Committee (IACUC)-approved guidelines and protocols. Compared with wild-type littermates (Ifi202WT), Ifi202KO animals developed normal B-cell populations and subpopulations, including marginal zone, B1 and transitional cells, as judged by appropriate B220, CD5, CD11b, CD23, CD21, immunoglobin (Ig)M and IgD staining of splenocytes and lymph node cells in flow cytometry (data not shown). Surprisingly, however, Ifi202KO B-cells exhibited largely normal apoptotic, proliferative and class switching/Ig secretion (of all Ig isotypes) phenotypes in vitro, in response to anti-IgM, anti-CD40 or lipopolysaccharide, in the presence or absence of IL-4, IFN- or TGF- (Fig. 1A–C and data not shown). Indeed, if anything, Ifi202KO B-cells appeared to demonstrate hyper-activity under some conditions, such as in response to anti-IgM þ IL-4 (Fig. 1A). In addition, Ifi202KO mice exhibited normal humoral responses to immunization with the Tdependent antigen, NP-KLH, as judged by total anti-NP IgM, IgG and IgG isotype analyses (Fig. 1D and data not shown). Thus, Ifi202a is grossly dispensable for B-cell functions and, if anything, suppresses B-cell function, at least as judged by B-cell proliferation and Ig production in vitro (Fig. 1A and C). These findings question the role of Ifi202 in the pathogenesis of lupus. In this sense, it is interesting to note that therapeutic interventions in some mouse lupus models fail to correlate with effects on Ifi202 activity and/or expression—e.g. Ifi202 levels are unaffected in NZB animals deficient in the type I IFN receptor despite their overall protection from disease [10]. The present findings suggest that at least under some conditions, Ifi202 actually suppresses B-cell activation and/or proliferation, perhaps through its previously described ability to suppress cellular growth and activity via Rb, E2F [4] and/or NF-B [5]. Thus the physiological functions of Ifi202 may be context-dependent, questioning its utility as a pathogenic target or disease biomarker in lupus. At the same time, it should be emphasized that the Ifi200 cluster does include additional members, such as Ifi203 and Ifi204, whose functions may be redundant with Ifi202. As such, although it seems clear from the present studies that Ifi202a is not necessary for normal B-cell homeostasis and function, making unlikely the possibility that its up-regulated expression accounts for the B-cell phenotype associated with the Nba2 locus [1], future studies will hopefully continue to explore the relative roles of the Ifi200 family members in lupus and B cells and perhaps other cell types, such as T-cells, which express Ifi202, with increased attention to the potentially overlapping and/or distinct roles of different family members.
Rheumatology key message Ifi202a-deficient mice lack any overt B-cell phenotype, suggesting that Ifi202a’s role in lupus, if present, is independent of B-cells, and/or that other members of the Ifi200 gene cluster regulate B-cell autoimmunity.
Acknowledgements We thank Peter Lengyel for the gracious provision of Ifi202adeficient mice.
Letters to the Editor
104 20 000
Ifi202WT Ifi202KO
B
Ifi202KO
Ifi202WT 20.2
20.1
Annexin V
Proliferation (cpm)
A
10 000
0.73
1.10
75.3
76.3 Propidium iodide
0 –
+IFNg
+IL–4
+TGFb
anti–IgM C
Ifi202WT Ifi202KO
750
anti–NP titre
Ig secreted (ng/mL)
20 000
D
1000
500 250 0 WT
KO
(no cytokine)
WT
KO
WT
+IL-4
KO
IFNg
10 000
0 0
5
10 15 20 Days after immunization
25
Lipopolysaccharide FIG. 1. Ifi202a is dispensable for B-cell homeostasis and function. Naı¨ve splenic B cells were isolated from Ifi202WT vs Ifi202KO mice via negative selection (Miltenyi Biotec, Auburn, CA, USA), and incubated in culture with anti-IgM (10 mg/ml), anti-CD40 (10 mg/ml) or lipopolysaccharide (LPS, 25 mg/ml) in the presence or absence of recombinant mouse IL-4 (10 ng/ml), IFN (100 ng/ml) and/or rhTGF1 (1 ng/ml). On day 2, cells were pulsed with 3H-thymidine, and harvested the next day to assess proliferation; or alternatively were analysed 24 h after stimulation by flow cytometry for apoptosis via Annexin V and propidium iodide staining (BD Pharmingen, San Diego, CA, USA). Supernatants were harvested on day 7–10 for assessment of Ig isotype secretion by ELISA (Southern Biotech). Representative (A) proliferation (in response to anti-IgM), (B) apoptosis (in response to anti-IgM) and (C) Ig secretion (IgG2b, in response to LPS) results for the conditions indicated. (D) Ifi202WT and Ifi202KO mice were immunized with 50 mg NP-KLH (Biosearch) emulsified in alum (Pierce). Sera were assessed on the days indicated for anti-hapten (NP)-specific IgG.
Funding: This work was supported in part by a grant from the National Institutes of Health (NIH) (R01 AI057571) to S.L.P. Disclosure statement: The authors have declared no conflicts of interest. M. R. GUBBELS BUPP, M. LI, A. PASHINE, D. AUD, S. L. PENG Roche Palo Alto, Palo Alto, CA, USA Accepted 21 August 2007 Correspondence to: S. L. Peng, 3431 Hillview Ave., M/S A2-259, Palo Alto, CA 94304, USA. E-mail:
[email protected] 1 Rozzo SJ, Allard JD, Choubey D et al. Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity 2001;15:435–43. 2 Landolfo S, Gariglio M, Gribaudo G, Lembo D. The Ifi 200 genes: an emerging family of IFN-inducible genes. Biochimie 1998;80:721–8. 3 Datta B, Li B, Choubey D, Nallur G, Lengyel P. p202, an interferon-inducible modulator of transcription, inhibits transcriptional activation by the p53 tumor suppressor protein, and a segment from the p53-binding protein 1 that binds to p202 overcomes this inhibition. J Biol Chem 1996;271:27544–55. 4 Choubey D, Li SJ, Datta B, Gutterman JU, Lengyel P. Inhibition of E2F-mediated transcription by p202. EMBO J 1996;15:5668–78. 5 Min W, Ghosh S, Lengyel P. The interferon-inducible p202 protein as a modulator of transcription: inhibition of NF-B, c-Fos, and c-Jun activities. Mol Cell Biol 1996;16:359–68. 6 Xin H, D’Souza S, Jorgensen TN et al. Increased expression of Ifi202, an IFN-activatable gene, in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis. J Immunol 2006;176:5863–70. 7 Pramanik R, Jorgensen TN, Xin H, Kotzin BL, Choubey D. Interleukin-6 induces expression of Ifi202, an interferon-inducible candidate gene for lupus susceptibility. J Biol Chem 2004;279:16121–7.
8 Hueber W, Zeng D, Strober S, Utz PJ. Interferon-alpha-inducible proteins are novel autoantigens in murine lupus. Arthritis Rheum 2004;50:3239–49. 9 Wang H, Chatterjee G, Meyer JJ et al. Characteristics of three homologous 202 genes (Ifi202a, Ifi202b, and Ifi202c) from the murine interferon-activatable gene 200 cluster. Genomics 1999;60:281–94. 10 Santiago-Raber ML, Baccala R, Haraldsson KM et al. Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice. J Exp Med 2003;197:777–88.
Rheumatology 2008;47:104–106 doi:10.1093/rheumatology/kem276 Advance Access publication 28 November 2007
Non-invasive quantification of popliteal cyst volumes by three-dimensional ultrasonography SIR, Popliteal cyst (Baker’s cyst) arises in the medial aspect of the popliteal fossa and results from fluid distension of the gastrocnemius-semimebranosus (GS) bursa, which is located between the muscles bearing the corresponding names [1]. The communication between the knee joint and the bursa, and the fluid dynamics of the cyst fluid contribute to the genesis of the popliteal cyst. Flexion of the knee causes the joint pressure to exceed bursal pressure and the valve-like connection between the joint and the bursa to open, whereas extension causes the tense GS muscle to close this ‘valve’ [2, 3]. In some pathological conditions, intraarticular pressure is so elevated that the fluid accumulates in and expands the bursal cavity. Few methods are available for the measurement of cyst volumes in arthritic patients and determining the cause of aggravated knee symptoms in patients who have had complicated popliteal
