Gene expression in mononuclear cells from patients with inflammatory bowel disease

Clinical Immunology 112 (2004) 247 – 257 www.elsevier.com/locate/yclim

Gene expression in mononuclear cells from patients with $ inflammatory bowel disease Elizabeth E. Mannick, a,b,* Joseph C. Bonomolo, b Ronald Horswell, a Jennifer J. Lentz, a Maria-Stella Serrano, b Adriana Zapata-Velandia, a,b Mariella Gastanaduy, a,b Jessica L. Himel, a,b Steven L. Rose, c John N. Udall, Jr., b Conrad A. Hornick, d and Zhiyun Liu b b

a Stanley S. Scott Cancer Center, New Orleans, LA 70112, USA Louisiana State University Inflammatory Bowel Disease Center of Excellence, New Orleans, LA 70112, USA c Department of Physiology, Louisiana State University, New Orleans, LA 70112, USA d Prometheus Laboratories, San Diego, CA 92121-4203, USA

Received 19 December 2003; accepted with revision 17 March 2004 Available online 4 June 2004

Abstract Objectives. Discovery of Nod2 as the inflammatory bowel disease 1 (IBD1) susceptibility gene has brought to light the significance of mononuclear cells in inflammatory bowel disease pathogenesis. The purpose of this study was to examine changes in gene expression in peripheral blood mononuclear cells in patients with untreated Crohn’s disease (CD) and ulcerative colitis (UC) as compared to patients with other inflammatory gastrointestinal disorders and to healthy controls. Methods. We used a 2400 gene cDNA glass slide array (MICROMAXk) to examine gene expression in peripheral blood mononuclear cells from seven patients with Crohn’s disease, five patients with ulcerative colitis, 10 patients with other inflammatory gastrointestinal disorders, and 22 age- and sex-matched controls. Results. Novel categories of genes differentially expressed in Crohn’s disease and ulcerative colitis patients included genes regulating hematopoietic cell differentiation and leukemogenesis, lipid raft-associated signaling, the actin cytoskeleton, and vesicular trafficking. Conclusions. Altered gene expression in mononuclear cells may contribute to inflammatory bowel disease pathogenesis. D 2004 Published by Elsevier Inc. Keywords: Crohn’s disease; Microarray; Mononuclear cell

Introduction Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn’s disease (CD), has been treated clinically for over 70 years, but the underlying cause and a definitive cure have yet to be found. IBD is thought to result from a complex interaction between environmental variables including microbial and dietary antigens, and polygenic genetic susceptibility. In the last 10 years, a global effort involving linkage disequilibrium mapping and positional cloning studies of multiply affected families with inflammatory bowel disease has uncovered a growing number of $ Supplementary data associated with this article can be found, in the online version, at doi:10.1016/S1521-6616(04)00104-4. * Corresponding author. Louisiana State University Inflammatory Bowel Disease Center of Excellence, 533 Bolivar Street, Room 409, New Orleans, LA 70112. Fax: +1-504-568-6888. E-mail address: [email protected] (E.E. Mannick).

1521-6616/$ - see front matter D 2004 Published by Elsevier Inc. doi:10.1016/j.clim.2004.03.014

susceptibility loci on chromosomes 1, 3, 4, 5, 10, 12, 16, 17, 22, and X [1– 8]. The first IBD susceptibility gene to be identified, Nod2, encodes an intracellular receptor for peptidoglycan, a component of bacterial cells walls. The downstream targets of Nod2 are RIP kinase and NF-kappa B. Three distinct polymorphisms in Nod2 (R702W, G908R, and 1007fs) are found with increased frequency in a subset of patients with CD and result in defective activation of NF kappa B in monocytes, intestinal epithelial cells, and Paneth cells exposed to peptidoglycan and lipopolysaccharide [9 –11]. The discovery that Nod2, a protein primarily expressed in mononuclear cells, could contribute to organ-specific disease placed mononuclear cells and innate immunity at the center of IBD pathogenesis for the first time. Recently, the Runx1 gene has been identified as a susceptibility gene for rheumatoid arthritis [12]. Runx 1 is a gene primarily expressed in hematopoietic cells that, when

248

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

fused to a variety of other genes, plays a role in myeloid malignancies. Polymorphisms in Runx1-binding sites in the genes encoding SLC22A4, SLCL9A3R1, and PDCD1 confer susceptibility to autoimmune diseases, rheumatoid arthritis, psoriasis, and systemic lupus erythematous [12 – 14]. These data further support the notion that hematopoietic cells may play a central role in chronic, inflammatory, and autoimmune conditions. Microarray analysis can complement genetic mapping strategies as a means of identifying signal transduction pathways and candidate genes in complex diseases. Microarray analysis has been used to study gene expression in mucosal biopsies from patients with ulcerative colitis, patients with CD, and healthy controls [15 – 18]. Genes known to be involved in the inflammatory response such as interleukins 1, 6, and 8, immunoglobulins, MHC Class II genes, matrix metalloproteinases, and complement family genes are upregulated in IBD. New categories of genes with altered expression in IBD have been detected, including defensin, Reg, Gro, and calcium-binding S100 genes. Two recent studies of colonic gene expression in patients with UC, patients with CD, and controls detected increased expression of colon cancer-related Wnt signaling and osteopontin genes in UC patients [17,18]. Here, we chose to focus on gene expression in PBMCs from patients with CD and UC because the Nod2 and Runx1 findings implicate mononuclear cells in the pathogenesis of IBD and other autoimmune disorders. We compared IBD patients to patients with other inflammatory gastrointestinal illnesses as well as to age- and sex-matched controls to identify genes that were specific to the disease and not specific to inflammation.

Methods Patients After obtaining informed consent, a total of 44 subjects were recruited into the study; 7 had untreated CD, 5 had untreated UC, 10 had another inflammatory gastrointestinal disorder (celiac disease, C. difficile colitis, H. pylori gastritis, acute food poisoning, disseminated mycobacterial infection, disseminated histoplasmosis, irritable bowel syndrome, colonic adenomatosis and diverticulosis, eosinophilic colitis, and sarcoidosis), and 22 were age- and sex-matched healthy controls. Diagnoses were made by a board-certified gastroenterologist based on a combination of clinical, radiographic, and endoscopic findings.

paque (Sigma, Kalamazoo, MI). RNA were extracted with Trisol reagent (Molecular Research Center, Cincinnati, OH) and purified with RNase-free DNase treatment (BD PharMingen, San Jose, CA) and phenol – chloroform – isoamyl extraction [19]. Quantity and integrity of RNA were assessed by spectrophotometry (DU640, Beckman Coulter, Fullerton, CA) and 1% agarose gel electrophoresis. Following the manufacturer’s protocol (Perkin-Elmer Life Sciences, Boston, MA), 2 Ag of RNA from each subject was labeled with either biotin (patient) or fluorescein (control) and converted to cDNA using reverse transcriptase in an iCycler (BioRad, Hercules, CA). DNA was purified by phenol – chloroform – isoamyl extraction [19]. After ethanol precipitation and denaturing at 90jC for 2 min, DNA was resuspended in hybridization buffer (Perkin-Elmer) and hybridized to a glass microarray slide spotted with 2400 cDNAs from known genes (MICROMAXk, Perkin-Elmer) at 65jC for 12 –16 h. After rinsing in stringent buffer, slides were incubated with secondary antibody (horseradish peroxidase (HRP)-conjugated streptavidin or anti-fluorescein HRP) and then with tyramide conjugated to either cyanine-3 (red) or cyanine-5 (green) dyes. Slides were read by confocal laser scanner and values for each gene in the array were expressed as a ratio of the red fluorescent intensity (patient) to green fluorescent intensity (control). Before data analysis, all arrays were median centered and log transformed. Thirty-five plant genes were included on the array as internal controls. The mean patient-to-control plant gene ratio for patients entered in this study was 1.08 F 0.5. Real-time PCR Primers for the genes of interest were designed using ABI Primer Express software (Applied Biosystems, San Diego, CA) and ordered from Integrated DNA Technologies, Inc. (IDT, Coralville, IA). Integrity of patient RNA was verified by 1% agarose gel electrophoresis and reverse transcription was performed on this RNA (60 ng to 2 Ag) using TaqMan RT Reagents and protocol (Applied Biosystems) in an ICycler (BioRad). Next, the primers were optimized using SYBR Green PCR Reagent and protocol (Applied Biosystems). A SYBR green master mix was prepared and aliquoted in 96-well Optical Reaction Plates (Applied Biosystems) with forward and reverse primers (50 – 100 nM). Using cDNA as a template for amplification, 5 Al of DNA was pipetted in each well. The 96-well plate was placed in an ABI Prism 7700 Sequence Detection System and analyzed using Sequence Detector v1.7. The relative gene expression was determined as fold change in patients compared to uninfected controls. Three cycles constituted a log10 change.

Microarrays Statistical analysis Each patient’s cDNA was hybridized on one slide with an age- and sex-matched control’s cDNA such that a total of 22 slides were evaluated. Six milliliters of whole blood was drawn from each subject and PBMCs separated on Histo-

To compare gene expression between patients with IBD and patients with other inflammatory disorders, t tests with bootstrapping and the Wilcoxon rank sum test were used.

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

Results are reported for genes for which either or both of the tests were significant at the P < 0.05 level. Comparisons between patients with CD and controls, patients with UC and controls, and patients with other GI illnesses and controls were made using the Wilcoxon matched-pairs signed-ranks test and are available as supplemental data on the web at http://www.clinimmsoc.org/journal).

Results and discussion Genes altered in expression in PBMCs from patients with Crohn’s disease as compared to patients with other GI inflammatory disorders A total of 28 genes were underexpressed and 24 genes were overexpressed in PBMCs from patients with CD as compared to patients with other inflammatory gastrointestinal disorders (Table 1). One limitation of the microarray analysis, in general, is the restriction of results to mRNA expression levels. Alterations in gene expression do not provide information on disease-related genetic polymorphisms or alterations in protein expression or functioning. Another limitation of the current study is the variability in the degree and type of inflammation in IBD patients and patients with other types of gastrointestinal inflammation. Because the data are derived from clinical specimens, it is impossible to match patients and controls with exactitude in regard to the amount of inflammation present. Thus, further studies will be necessary to validate the biological relevance of the genes detected in these and other microarray studies. Despite the inherent limitations of the methodology, several interesting categories of genes were identified in this study that could play a role in disease pathogenesis based on existing knowledge from animal and human studies of IBD. Genes encoding transcription factors that play a role as transcriptional repressors or regulators of cellular differentiation were differentially expressed (Table 1A). Expression of the transforming growth factor beta-induced factor (TGIF) gene, a member of the TALE homeobox domain family, was increased [20]. TGIF is induced by transforming growth factor (TG) beta and encodes a transcriptional corepressor that inhibits 9-cis-retinoic acid-induced transcriptional activation by RXR alpha of the retinoic acid responsive element [21]. It also antagonizes SMAD2 functioning downstream of TGF-beta by binding SMAD2 and histone deacetylases [20]. If changes in gene expression result in parallel changes in protein expression and activation, the observed upregulation of TGIF is of potential significance to CD pathogenesis for two reasons. First, TGF-beta and retinoic acid regulate myeloid differentiation, a process that may be involved in Nod2 pathogenesis in CD [22]. Second, Smad2 signaling mediates TGF-beta suppression of interferon-gamma and TNF-alpha signaling [23]. If Smad 2 is rendered nonfunctional by an overexpression of TGIF, then TGF-beta is

249

crippled in its ability to terminate immune responses. Defective TGF-beta signaling has long been suspected to contribute to CD pathogenesis [24]. In contrast to TGIF, expression of the competing TALE family member meis2 was repressed (Table 1A) [25]. Like TGIF, meis2 plays an inhibitory role in myeloid differentiation. Unlike TGIF, meis2 expression is upregulated by retinoic acid [26]. Cell culture studies indicate that TGIF can directly compete with meis2 for binding to DNA and suppress meis2-induced transcriptional activation [25]. It is therefore possible to postulate that overexpression of TGIF could result in a suppression of meis2 expression in CD. Perhaps not surprisingly, the downstream gene target of meis2, PAX6, was underexpressed as well [27]. Pax6 is a homeobox domain gene that regulates development in concert with HoxB1 and Pbx transcription factors [28]. The MLL gene, a transcription factor directly linked to leukemogenesis, was underexpressed (Table 1A) [29]. The MLL gene encodes a trithorax homolog that acts as a transcriptional repressor with histone deacetylases [30]. Fusion of MLL with many different genes results in acute myeloid leukemia [31]. Interestingly, several MLL fusion partners or their binding partners were differentially expressed in CD and UC. These include eps15 (Table 1D) and sorting nexin 2 (Table 2C), which binds formin-binding protein 17 [30,32]. Although eps15 is known to be involved in clathrin-mediated endocytosis, it has been localized to the nucleus and can act as a transcriptional activator, perhaps explaining its oncogenic effects [33]. Two genes that participate in NF kappa B activation were overexpressed, Rel proto-oncogene (c-rel) and ubiquitinconjugating enzyme E2 variant (Table 1A) [34,35]. Excessive activation of the transcription factor, NF-kappa B, plays a role in inflammation and cancer, including hematologic malignancies [36]. NF kappa B is both a transcriptional activator and the downstream target of Nod2. CD-associated Nod2 mutations lead to defective NF kappa B activation in response to peptidoglycan [10,37]. In addition, the leukemia-associated genes RP58 and TLS/CHOP were overexpressed (Table 1A). RP58 is a transcriptional corepressor and TLS/CHOP is an oncogene that inhibits differentiation in myeloid cells [38,39]. In addition to transcription factors, cytoplasmic genes that play a role in cellular differentiation were differentially expressed in patients with CD. These included two underexpressed genes, Pig10, a retinoic acid-induced gene associated with p53, apoptosis, and differentiation, and Placenta (Diff48) [40,41] (Table 1B). The significance of the differential expression of transcription factor genes involved in differentiation, retinoic acid signaling, and leukemogenesis is unknown. However, Runx-1, which may prove to be central to the pathogenesis of autoimmunity, is directly involved in differentiation, retinoic acid signaling, and leukemogenesis [42]. Moreover, clinical experience suggests that CD patients have a higher incidence of myelodysplasia and myeloid leukemia [43]. It

250

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

Table 1 Gene expression in patients with Crohn’s disease vs. other GI disorders Genes by category (chromosomal location)

z# (Average fold change F SE, P value)

A. DNA- and RNA-binding or transcription factors RP58 (ZNF238) (1q44) z (1.46 F 0.23 vs. 0.92 F 0.13, P = 0.025) Infant brain unknown product z (1.40 F 0.25 vs. 0.85 F 0.08, (origin recognition complex, P = 0.025) subunit 3) (6q14.3 – q16.1) TGIF protein (tale homeobox z (2.07 F 0.57 vs. 1.01 F 0.22, protein) (18p11.3) P = 0.040) Putative cyclin G1 interacting z (1.26 F 0.08 vs. 0.98 F 0.08, protein (zinc finger, HIT P = 0.016) domain containing 1) (7q22.1) Rel proto-oncogene (2p13 – p12) z (1.49 F 0.21 vs. 0.84 F 0.11, P = 0.010) Ubiquitin conjugating enzyme E2 z (1.57 F 0.37 vs. 1.40 F 0.35, variant (DNA-binding protein/ P = 0.025) CROC1B) (20q13.2) KIAA0134 gene (DEAD box # (0.73 F 0.18 vs. 1.29 F 0.18, polypeptide 34) (19q13.3) P = 0.040) LAK-1 (5p15) # (0.65 F 0.10 vs. 1.04 F 0.14, P = 0.040) MLL (myeloid/lymphoid # (0.69 F 0.11 vs. 1.35 F 0.21, leukemia) (11q23) P = 0.008) Meis 2 (msg1-related gene # (0.77 F 0.14 vs. 1.26 F 0.18, 1/meis1 homolog) (15q13.3) P = 0.030) Paired box gene (PAX6) # (0.84 F 0.14 vs. 1.86 F 0.33, homologue (11p13) P = 0.032) TLS/CHOP, hybrid gene z (1.27 F 0.16 vs. 0.94 F 0.19, (16p11.2) P = 0.040) B. Differentiation Pig10 (PIG10) (5q13) Placenta (Diff48) (6p22.3 – p21.32) C. Cytoskeletal genes Microfibrillar-associated protein 1 (15q15 – q21) KIAA0373 gene (12q21.33) KIAA0389 gene (Myosin VI) (6q13) 2V,3V-Cyclic nucleotide 3V-phosphodiesterase (17q21) Cytoplasmic dynein intermediate chain isoform IC-2 (7q21.3 – q22.1) Desmin (2q35) Slow skeletal troponin C (TnC) (3p21.3 – p14.3) Striatin (2p22 – p21)

D. Vesicular trafficking Alpha SNAP (19q13.33) Cytohesin2/plekstrin homology, Sec7 and coiled-coil domains 2 (19q13.33) Epidermal growth factor receptor substrate (eps15) (1p32)

# (0.76 F 0.13 vs. 1.47 F 0.21, P = 0.028) # (0.62 F 0.14 vs. 1.34 F 0.30, P = 0.032)

z (1.19 F 0.07 vs. 0.85 F 0.13, P = 0.032) z (1.96 F 0.44 vs. 0.91 F 0.10, P = 0.005) z (1.54 F 0.31 vs. 0.84 F 0.11, P = 0.008) # (0.58 F 0.13 vs. 1.80 F 0.47, P = 0.008) # (0.86 F 0.12 vs. 1.30 F 0.14, P = 0.019) # (0.61 F 0.20 vs. 0.96 F 0.08, P = 0.040) # (0.82 F 0.09 vs. 2.03 F 0.40, P = 0.004) # (0.81 F 0.12 vs. 1.11 F 0.08, P = 0.051)

Table 1 (continued) Genes by category (chromosomal location)

z# (Average fold change F SE, P value)

D. Vesicular trafficking KIAA0064 gene (sorting nexin 17) Mosaic protein LR11 (sorLA) (11q23.2 – q24) ORF, Xq terminal portion (lysosomal H+ transporting ATPase/ATP6IP1) (Xq28)

# (0.82 F 0.12 vs. 1.36 F 0.25, P = 0.025) # (0.76 F 0.19 vs. 1.33 F 0.21, P = 0.032) # (0.82 F 0.19 vs. 1.32 F 0.18, P = 0.040)

E. Inflammation/intracellular signal transduction Enhancer of filamentation (HEF1) z (1.89 F 0.43 vs. 1.16 F 0.25, (Nedd 9/cas-like docking) P = 0.032) (6p25 – p24) Epidermal growth factor z (1.36 F 0.18 vs. 0.90 F 0.09, receptor-binding protein P = 0.036) GRB2 (17q24 – q25) HYL tyrosine kinase z (1.75 F 0.38 vs. 0.91 F 0.09, (megakaryocyte associated P = 0.042) tyrosine kinase) (19p13.3) Ceramide glucosyltransferase z (1.21 F 0.08 vs. 0.86 F 0.16, (9q31) P = 0.026) Neuropeptide Y receptor Y1 z (1.75 F 0.38 vs. 1.01 F 0.15, (NPYY1) (4q31.3 – q32) P = 0.032) Surface antigen (flotillin 2) z (1.32 F 0.26 vs. 2.01 F 0.78, (17q11 – q12) P = 0.040) TLS/CHOP, hybrid gene z (1.27 F 0.16 vs. 0.94 F 0.19, (16p11.2) P = 0.040) Type 1 neurofibromatosis protein z (1.45 F 0.18 vs. 1.03 F 0.13, (17q11.2) P = 0.040) Endothelial-monocyte activating # (0.86 F 0.19 vs. 1.53 F 0.20, polypeptide II (SCYE1) P = 0.025) (4q25) Heparin-binding EGF-like growth # (0.65 F 0.11 vs. 1.45 F 0.42, factor (diptheria toxin) (5q23) P = 0.032) SHPS-1 (20p13) # (0.87 F 0.24 vs. 1.68 F 0.24, P = 0.040) Aspartyl beta-hydroxylase # (0.82 F 0.13 vs. 1.56 F 0.29, (8q12.1) P = 0.024) 43 kDa inositol polyphosphate # (0.78 F 0.13 vs. 1.46 F 0.24, 5-phosphatase (INPP5A) P = 0.038) (10q26.3) F. Oxidative stress Natural killer cell enhancing factor (NKEFB) (periredoxin 2) (19p13.2) Glutathione peroxidase 1 (3p21.3) TorsinA (DYT1) (9q34)

z (1.34 F 0.13 vs. 0.90 F 0.13, P = 0.034) z (1.79 F 0.46 vs. 1.03 F 0.08, P = 0.040)

G. Other Chromosome 3p21.1 gene sequence (3p21.31) Putative transmembrane protein precursor (B5) (integral membrane protein 1) (11q23.3) RNaseP protein p30 (RPP30) (10q23.32 – q23.33 Bleomycin hydrolase (17q11.2)

# (0.44 F 0.08 vs. 1.13 F 0.25, P = 0.040)

Citrate transporter protein (SLC25A1) (22q11)

z (1.75 F 0.49 vs. 0.80 F 0.09, P = 0.025) # (1.79 F 0.76 vs. 1.88 F 0.64, P = 0.046) # (0.82 F 0.11 vs. 1.26 F 0.13, P = 0.030)

z (1.67 F 0.27 vs. 1.16 F 0.15, P = 0.046) z (1.99 F 0.46 vs. 1.23 F 0.30, P = 0.051) z (1.49 F 0.21 vs. 0.80 F 0.08, P = 0.000) # (0.77 F 0.15 vs. 1.53 F 0.33, P = 0.047) # (0.78 F 0.13 vs. 1.28 F 0.17, P = 0.030)

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257 Table 1 (continued) Genes by category (chromosomal location)

z# (Average fold change F SE, P value)

G. Other Heparan sulfate-6-sulfotransferase (2q21) Platelet-type phosphofructokinase (10p15.3 – p15.2) Sorbitol dehydrogenase gene (15q15.3)

# (0.81 F 0.20 vs. 1.52 F 0.23, P = 0.043) # (1.23 F 0.36 vs. 1.37 F 0.18, P = 0.040) # (0.81 F 0.19 vs. 2.03 F 0.65, P = 0.032)

also suggests that the use of retinoic acid derivatives can trigger IBD in susceptible individuals, pointing to a possible dysregulation of retinoic acid signaling in CD [44]. Genes related to the cellular cytoskeleton were also differentially expressed in CD (Table 1C). Several myofibrillar protein genes were overexpressed in CD: myosin VI, a gene involved in endocytosis and secretion in mononuclear cells; microfibrillar-associated protein; and the myosin heavy chain-like gene, KIAA0373 [45,46]. In contrast, expression of the actin- and tropomyosin-binding gene, slow skeletal troponin C was decreased [47]. In addition, two genes involved in microtubule assembly were downregulated in CD, 2V3V-cyclic nucleotide 3V-phosphodiesterase and cytoplasmic dynein intermediate chain, as was the cyclosporin Ainducible intermediate filament gene, desmin [48 –50]. Whether these changes in the expression of cytoskeletal genes translate into altered cytoskeletal architecture and motility in patients’ PBMCs remains to be seen. It is potentially relevant, however, that the microtubule organizing center is the focal point of the T-cell macrophage immune synapse and disruption of microtubules inhibits LPS-induced TNF production in monocytes and macrophages [51]. In addition, disruption of the actin cytoskeleton appears to be central to the pathology of the spontaneous colitis seen in Wiskott Aldrich syndrome protein (WASP) knockout mice [52]. Thus, it is conceivable that defects in this pathway contribute to CD pathogenesis in humans. Another functionally related group of differentially expressed genes in CD were vesicular trafficking genes (Table 1D). Two genes involved in trans-Golgi transport, alpha SNAP and cytohesin 2, were induced [53,54]. Three genes linked to receptor-mediated endocytosis, eps15, the sortilin related receptor 1 (SORL1), and sorting nexin 17, were underexpressed [55 –57]. In addition, the lysosomal H+ transporting ATPase gene, involved in endosomal and lysosomal acidification, was underexpressed [58]. Eps 15 is a substrate of growth factor receptors that regulates clathrinmediated endocytosis [55]. Sorting nexin 17 internalizes Pselectin [57]. Based on these results, it is possible to hypothesize that there may be a defect in endocytosis in mononuclear cells from patients with CD, but the functional significance of such a defect remains unclear. Failure to internalize receptors and sort them to early endosomes (or excessive recycling of receptors back to the cell surface in the case of UC) can prolong receptor-mediated intracellular

251

signaling. In T-cells, CD4 is internalized by clathrin-mediated apoptosis [59], and its defective internalization or excessive cell surface recycling could lead to a prolongation of T-cell receptor-mediated signaling in IBD. Likewise, failure to internalize sorting nexin 17 could be hypothesized to result in prolonged P-selectin signaling with resultant inflammation in CD. Table 2 Ulcerative colitis (n = 5) vs. other GI disorders (n = 10) Genes by category (chromosomal location) A. DNA and RNA binding Transducin (beta) like 1 protein (Xp22.3) U2 small nuclear RNA-associated BW antigen (SNRPB2) (20p12.2 – p11.22) RNA polymerase II elongation factor SIII, p15 subunit (TCEB1) (8q13.3) Zinc finger protein (BCL-6)

B. Cytoskeletal genes Ankyrin G (ANK-3) (10q21) Profilaggrin (1q21)

C. Vesicular trafficking AP-3 complex delta subunit (19p13.3) ARL1 (12q23.3) Rab11a GTPase (15q21.3 – q22.31) Subunit of coatomer complex (COPB2) (3q23) Sorting nexin 2 (SNX2) (5q23)

#z(Average fold change F SE, P value) z (1.74 F 0.69 vs. 1.04 F 0.20, P = 0.023) z (2.71 F 0.58 vs. 1.42 F 0.22, P = 0.023) # (0.90 F 0.12 vs. 1.22 F 0.09, P = 0.036) # (0.80 F 0.13 vs. 1.18 F 0.13, P = 0.050)

# (0.68 F 0.09 vs. 1.38 F 0.38, P = 0.039) # (0.65 F 0.34 vs. 1.17 F 0.28, P = 0.039)

z (1.74 F 0.29 vs. 0.98 F 0.11, P = 0.024) z (3.02 F 0.95 vs. 1.06 F 0.24, P = 0.007) z (2.81 F 0.42 vs. 1.51 F 0.28, P = 0.023) z (3.56 F 1.65 vs. 1.08 F 0.09, P = 0.003) # (0.53 F 0.08 vs. 1.09 F 0.09, P = 0.002)

D. Inflammation or intracellular signal transduction Caveolin (7q31.1) z (1.70 F 0.21 vs. 0.98 F 0.16, P = 0.038) Striatin (2p22 – p21) z (1.88 F 0.31 vs. 1.11 F 0.08, P = 0.039) Osteopontin (4q21 – q25) # (0.73 F 0.15 vs. 1.16 F 0.16, P = 0.039) E. Oxidative stress Glutathione-S-transferase homolog (omega) (10q25.1) Selenoprotein W (hSelW) (19q13.3) F. Other Pancreatic beta-cell glucokinase (7p15.3 – p15.1) Sodium/potassium-transporting ATPase beta-3 subunit (3q22 – q23) Methionine aminopeptidase (4q23)

z (5.33 F 2.85 vs. 1.53 F 0.24, P = 0.039) z (1.70 F 0.07 vs. 1.15 F 0.09, P = 0.009)

z (1.63 F 0.52 vs. 0.81 F 0.07, P = 0.013) z (2.86 F 0.21 vs. 1.34 F 0.27, P = 0.022) # (0.44 F 0.17 vs. 1.20 F 0.28, P = 0.032)

252

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

IBD involves dysregulation of intracellular signaling pathways in T-lymphocytes and monocytes, but the exact nature of the signaling defect remains unknown. Recent data from human and mouse studies suggest that defects in the STAT3 signaling pathway may play a role in CD pathogenesis [60,61]. Nod2 data point to TNF-alpha and NF kappa B signaling pathways as well [37]. Finally, Runx1 data obtained from genetic studies of psoriasis suggest a defect in intracellular signaling in lipid rafts involving SLC9A3R1 (NHERF1), an ezrin-radixin-moesin family member and Csk binding protein [13]. Lipid rafts and caveolae containing molecules such as SLC9A3R1, Csk, Csk binding protein, and Src kinases regulate adhesion, migration, and the cytoskeleton as well as signaling downstream from the T-cell and B-cell receptors [62]. Interestingly, our microarray data show altered expression of signal transduction molecules in all three of these pathways (Table 1E). In CD (Table 1E), Grb2, an adaptor molecule that binds SOCS-1 and negatively regulates Stat3 in response to growth factor signaling, was overexpressed [63]. Similarly, the heparin-binding EGF-like growth factor, an inducer of growth factor-mediated signaling in response to bacterial lipoteichoic acid, was underexpressed in CD [64]. Endothelial monocyte-activating polypeptide II, a chemokine gene that activates ERK kinases and NF-kappa B, was suppressed [65]. Conversely, type I neurofibromatosis protein, a negative regulator of RAS and ERK signaling, was induced [66]. Together, these findings point to a possible downregulation of STAT3 and ERK signaling in PBMCs in CD. This is intriguing because mice with a targeted disruption of Stat3 in monocytes develop IBD [60]. Moreover, dysregulated ERK signaling in T-cells has been shown to play a role in defective tolerance and lupus pathogenesis [67]. In addition, ERK activation is necessary for Stat3 activation in response to G-protein-coupled receptor stimulation [68]. SHPS-1, a gene that negatively regulates LPS-stimulated TNF-alpha secretion through the activation of PI3-kinase, was underexpressed (Table 1E) [69]. SHPS-1 also renders dendritic cells insensitive to IL-12 signaling by inhibiting their maturation [70]. It is possible to speculate that the excessive TNF alpha secretion and IL-12 signaling observed in CD could result from underexpression of SHPS-1. However, further experiments will be required to test this hypothesis. In CD (Table 1E), the upregulated gene, enhancer of filamentation (Cas-L, HEF1), encodes a TGF-beta protein that is tyrosine phosphorylated by c-src kinases downstream from the TCR and BCR [71]. HEF1 positively regulates cellular migration through interactions with focal adhesion kinase (FAK) [72]. In contrast, Hyl, a Csk-like protein that negatively regulates c-src kinases and T-cell proliferation, was upregulated in CD as well. Hyl phosphorylates and inactivates the c-src family member, lyn [73]. This finding is of potential interest to IBD pathogenesis because lyn/ mice develop autoimmunity and B-cell hyperresponsiveness [74]. Flotillin 2, a signaling molecule contained in lipid rafts

that colocalizes with the TCR and microtubule organizing center, and the lipid raft-associated ceramide glycosyltransferase were overexpressed in CD [75,76]. Striatin, a caveolin-binding protein that binds sorting nexin 2 and eps15, was underexpressed [77,78]. Two genes that regulate calcium storage and mobilization were inhibited in CD (Table 1E), aspartyl beta hydroxylase and inositol polyphosphate 5 phosphatase (INPP5A) [79,80]. Patients with CD also suffer from an increased incidence of small and large intestinal cancers [81]. Interestingly, mice with a targeted disruption of aspartyl beta hydroxylase have an increased incidence of intestinal neoplasia [82]. Three genes induced by oxidant stress that perform protective roles were differentially expressed in CD (Table 1F); the natural killer cell enhancing factor (peroxiredoxin) gene was induced but the glutathione peroxidase 1 and torsinA genes were suppressed [83 – 85]. Other differentially expressed genes in patients with CD (Table 1G) included the overexpressed autoantigen gene, RNase P protein P30, and the underexpressed metabolic genes, platelet type phosphofructokinase, sorbitol dehydrogenase, and citrate transporter [86 – 89]. Genes altered in expression in PBMCs from patients with ulcerative colitis as compared to patients with other GI inflammatory disorders A total of 19 genes were differentially expressed in PBMCs from patients with UC compared to patients with other GI illnesses; 7 were underexpressed and 12 were overexpressed (Table 2). A number of DNA- and RNA-binding molecules were differentially expressed (Table 2A). The transducin beta like 1 gene was upregulated, encoding a transcriptional repressor that forms a complex with NCOR1 and histone deacetylase and participates in beta catenin destruction in response to genotoxic damage [90,91]. UC patients have an increased risk of developing colon cancer and lymphoma [81]. One of the mechanisms of colon carcinogenesis is defective beta catenin degradation leading to the nuclear translocation of free beta catenin. Increased transducin beta-like 1 expression could be postulated to act as a defense against carcinogenesis in UC. Expression of the proto-oncogene, Bcl-6, which is involved in the transcriptional repression of lymphocyte activation genes, was also decreased in UC. Bcl-6/ mice develop multiorgan system inflammation with eosinophils and an exaggerated Th2 responsiveness [92]. It is interesting to hypothesize that this mechanism may be involved in UC pathogenesis. The RNA polymerase II elongation factor SIII was also underexpressed in UC [93]. The RNA-associated autoantigen, U2 small nuclear RNA-associated B antigen (SNRPB2), was overexpressed [94]. Two cytoskeletal genes were underexpressed in UC, ankyrin 3, a molecule linking integral membrane proteins to the actin-spectrin cytoskeleton, and profilaggrin, an intermediate filament protein (Table 2B) [95,96]. Intrigu-

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

ingly, profilaggrin is a major rheumatoid arthritis autoantigen [96]. Spectrin –ankyrin complexes have been shown to inhibit membrane expression of CD45 in lymphocytes and T-cell activation [97]. This is interesting because patients with ulcerative colitis have higher percentages of CD45+ lymphocytes in colonic mucosa [98]. Vesicular trafficking molecules were differentially expressed in UC patients (Table 2C). In UC, there was an increase in Rab11a expression, a molecule that increases recycling of receptors from the endosome to the cell surface [99]. It is interesting to speculate whether the net effect of increased expression of a gene involved in recycling of receptors back to the cell surface would be similar to that of decreased expression of molecules involved in endocytosis of cell surface receptors observed in CD. As in CD, genes encoding molecules involved in transport to and from the Golgi (Table 2C) were overexpressed in UC. These included ARL1 and COPB2 [100,101]. In UC, the significantly underexpressed sorting nexin 2 gene, which mediates ER to Golgi transport, was the exception [102]. These findings imply a possible increase in secretion as might be expected in activated mononuclear cells. Interestingly, COPB2 is an autoantigen in scleroderma and ARL1 transports the autoantigen Golgin to the Golgi [100,101]. AP-3 complex delta, a gene promoting Golgi to cell membrane transport of proteins, was overexpressed in UC (Table 2C) [103]. This is interesting because mutations in the AP-3 complex delta gene cause a Hermansky –Pudlak-like syndrome in mice [104]. Hermansky– Pudlak syndrome in humans significantly elevates the risk of developing IBD [105]. Differentially expressed membrane-bound intracellular signaling molecules (Table 2D) included the upregulated gene caveolin, a component of lipid rafts, and striatin, a caveolin-binding molecule [77,106]. Caveolin is a src tyrosine kinase substrate that plays a role in negative feedback of src kinase signaling by binding Csk, a src inhibitor [106]. Csk binds Csk-binding protein and inhibits TCR signaling as well as src signaling in monocytes [107]. Intriguingly, striatin was upregulated in UC but downregulated in CD (Table 1C). As mentioned above, striatin binds sorting nexin 2 (underexpressed in UC) and eps15 (underexpressed in CD), two molecules that play an important role in vesicular trafficking. Moreover, sorting nexin 2 and eps15 bind MLL, providing MLL with a link to epidermal growth factor receptor signaling [30,32,78]. What these findings imply for IBD pathogenesis remains unknown, however. Surprisingly, osteopontin, a tumor-suppressor gene that is overexpressed in both UC colonic tissue and in colon cancer, was underexpressed in PBMCs from patients with UC in this study [18,108]. Because expression of osteopontin has been associated with Th1 responses, decreased expression in PBMCs in UC could simply reflect a Th2 polarization of peripheral T-cells [109]. Two genes induced by oxidative stress were upregulated in UC patients: glutathione S transferase omega and sele-

253

Table 3 Comparison of average fold change (D) in selected genes in PBMCs in IBD vs. other GI disorders by real-time PCR Crohn’s disease (n = 5) vs. other GI (n = 5) Gene

Microarray (Average fold D)

Real-time PCR (Average fold D)

Eps15 MLL SHPS-1 TGIF

2.6 1.9 1.9 2.0

9.1 1.3 4.4 1.2

noprotein W (Table 2E) [110,111]. The diabetes susceptibility gene, pancreatic glucokinase, and the ion channel, sodium-potassium ATPase beta 3, were overexpressed [112,113]. Methionine aminopeptidase, a gene involved in cleavage of the initiator methionine from proteins, was downregulated (Table 2F) [114]. Real-time confirmation of selected genes SYBR green data were obtained for selected differentially expressed genes in CD patients to confirm microarray findings in a subset of CD patients for whom additional RNA was available. The direction of change was the same for all genes tested although microarray results underexpressed the magnitude of the change found by real-time PCR in the case of eps15 and SHPS-1 (Table 3). Conclusions The identification of Nod2 as the IBD1 susceptibility gene and Runx1 as an autoimmunity susceptibility gene underscores the significance of mononuclear cells in the pathogenesis of inflammatory bowel disease and autoimmunity in general [9 –14]. Using microarray analysis, we have detected differential expression of a wide variety of genes in PBMCs from patients with CD as compared to normal controls and to patients with UC. These include transcription factor genes involved in differentiation and leukemogenesis, lipid raft signaling genes, cytoskeletal genes, and vesicular trafficking genes. Future studies will address the role of some of these genes in IBD pathogenesis.

Acknowledgments This study was supported by a First Award from the Crohn’s and Colitis Foundation of America, as well as unrestricted grants from the Toler Foundation, the Solomon family, and Prometheus Laboratories to E.E.M.

References [1] J.P. Hugot, P. Laurent-Puig, C. Gower-Rousseau, et al., Mapping of a susceptibility locus for Crohn’s disease on chromosome 16, Nature 379 (1996) 821 – 823.

254

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

[2] J.H. Cho, D.L. Nicolae, R. Ramos, et al., Linkage and linkage disequilibrium in chromosome band 1p36 in American Chaldeans with inflammatory bowel disease, Hum. Mol. Genet. 9 (2000) 1425 – 1432. [3] J. Hampe, N.J. Lynch, S. Daniels, et al., Fine mapping of the chromosome 3p susceptibility locus in inflammatory bowel disease, Gut 48 (2001) 191 – 197. [4] J. Satsangi, M. Parkes, E. Louis, et al., Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12, Nat. Genet. 14 (1996) 199 – 202. [5] J. Hampe, S. Schreiber, S.H. Shaw, et al., A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort, Am. J. Hum. Genet. 64 (1999) 808 – 816. [6] J. Hampe, S.H. Shaw, R. Saiz, et al., Linkage of inflammatory bowel disease to human chromosome 6p, Am. J. Hum. Genet. 65 (6) (1999 Dec) 1647 – 1655. [7] J.D. Rioux, M.S. Silverberg, M.J. Daly, et al., Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci, Am. J. Hum. Genet. 66 (2000) 1863 – 1870. [8] R.H. Duerr, M.M. Barmada, L. Zhang, et al., High-density genome scan in Crohn disease shows confirmed linkage to chromosome 14q11 – 12, Am. J. Hum. Genet. 66 (6) (2000 Jun.) 1857 – 1862. [9] J.P. Hugot, M. Chamaillard, H. Zouali, et al., Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease, Nature 411 (2001) 599 – 603. [10] Y. Ogura, D.K. Bonen, N. Inohara, et al., A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease, Nature 411 (2001) 603 – 606. [11] Y. Ogura, S. Lala, W. Xin, E. Smith, T.A. Dowds, F.F. Chen, E. Zimmermann, M. Tretiakova, J.H. Cho, J. Hart, J.K. Greenson, S. Keshay, G. Nunez, Expression of NOD2 in Paneth cells: a possible link to Crohn’s ileitis, Gut 52 (2003) 1591 – 1597. [12] S. Tokuhiro, R. Yamada, X. Chang, A. Suzuki, Y. Kochi, T. Sawada, S. Masakatsu, M. Nagasaki, M. Ohtsuki, M. Onon, H. Furukawa, M. Nagashima, S. Yoshino, A. Mabuchi, A. Sekine, S. Saito, A. Takahashi, T. Tsunoda, Y. Nakamura, K. Yamamoto, An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transorter, is associated with rheumatoid arthritis, Nat. Genet., (electronic publication ahead of print). [13] C. Helms, L. Cao, J.G. Krueger, E.M. Wijsman, F. Chamian, D. Gordon, M. Heffernana, J.A. Wright Daw, J. Robarge, J. Ott, P.-Y. Kwok, A. Menter, A.M. Bowcock, A putative RUNX1 binding site variant between SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis, Nat. Genet., (electronic publication ahead of print). [14] L. Prokunina, C. Castillejo-Lopez, F. Oberg, I. Gunnarsson, L. Berg, V. Magnusson, A.J. Brookes, D. Tentler, H. Kristiansdottir, G. Grondal, A.I. Bolstad, E. Svenungsson, I. Lundberg, G. Sturfelt, A. Jonssen, L. Truedsson, G. Lima, J. Alcocer-Varela, R. Jonsson, U.B. Gyllensten, J.B. Harley, D. Alarcon-Segovia, K. Steinsson, M.E. Arcon-Riquelme, A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematous in humans, Nat. Genet. 32 (2002) 666 – 669. [15] I.C. Lawrence, C. Fiocchi, S. Chakravarti, Ulcerative colitis and Crohn’s disease: distinctive gene expression profiles and novel susceptibility candidate genes, Hum. Mol. Genet. 10 (2001) 445 – 456. [16] B.K. Dieckgraefe, W.F. Stenson, J.R. Korzenik, et al., Analysis of mucosal gene expression in inflammatory bowel disease by parallel oligonucleotide arrays, Physiol. Genomics 4 (2000) 1 – 11. [17] S.M. Uthoff, M.R. Eichenberger, R.K. Lewis, et al., Identification of candidate genes in ulcerative colitis and Crohn’s disease using cDNA array technology, Int. J. Oncol. 19 (2001) 803 – 810. [18] H. Masuda, Y. Takahashi, S. Asai, T. Takayama, Distinct gene expression of osteopontin in patients with ulcerative colitis, J. Surg. Res. 111 (2003) 85 – 90. [19] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Plainview, NY; 1989, pp. E.3 – E.4.

[20] D. Wotton, P.S. Knoepfler, C.D. Laherty, R.N. Eisenman, J. Massague, The SMAD transcriptional corepressor TGIF recruits mSin3, Cell Growth Differ. 12 (2001) 457 – 463. [21] E. Bertolino, B. Reimund, D. Wildt-Perinic, R.G. Clerc, A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif, J. Biol. Chem. 270 (1995) 31178 – 31188. [22] O. Gutierrez, C. Pipaon, N. Onohara, A. Fontalba, Y. Ogura, F. Prosper, G. Nunez, J.L. Fernandez-Luna, Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation, J. Biol. Chem. 277 (2002) 41701 – 41705. [23] K. Sumiyoshi, A. Nakao, Y. Setoguchi, R. Tsuboi, K. Okumura, H. Ogawa, TGF-beta/Smad signaling inhibits IFN-gamma and TNFalpha-induced TARC (CCL17) production in HaCaT cells, J. Dermatol. Sci. 31 (2003) 53 – 58. [24] G. Monteleone, A. Kumberova, N.M. Croft, C. McKenzie, H.W. Steer, T.T. MacDonald, Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease, J. Clin. Invest. 108 (2001) 601 – 609. [25] Y. Yang, C.K. Hwang, U.M. D’Souza, S.H. Lee, E. Junn, M.M. Mouradian, Three-amino acid extension loop homeodomain proteins Meis2 and TGIF differentially regulate transcription, J. Biol. Chem. 275 (2000) 20734 – 20741. [26] N. Mercader, E. Leonardo, M.E. Piedra, A.C. Martinez, M.A. Ros, M. Torres, Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes, Development 127 (2000) 3961 – 3970. [27] X. Zhang, A. Friedman, S. Heaney, P. Purcell, R.L. Maas, Meis homeoproteins directly regulate Pax6 during vertebrate lens morphogenesis, Genes Dev. 16 (2002) 2097 – 2107. [28] I. Mikkola, J.A. Bruun, T. Holm, T. Johansen, Superactivation of Pax6-mediated transactivation from paired domain-binding sites by DNA-independent recruitment of different homeodomain proteins, J. Biol. Chem. 276 (2001) 4109 – 4118. [29] S. Ziemin-van der Poel, N.R. McCabe, H.J. Gill, R. Espinosa, Y. Patel, A. Harden, P. Rubinelli, S.D. Smith, M.M. Le Beau, J.D. Rowley, M.O. Diaz, Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias, Proc. Natl. Acad. Sci. 88 (1991) 10735 – 10739. [30] Z.B. Xia, M. Anderson, M.O. Diaz, N.J. Zeleznik-Le, MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 8342 – 8347. [31] C.W. So, M. Lin, P.M. Ayton, E.H. Chen, M.L. Cleary, Dimerization contributes to oncogenic activation of MLL chimeras in acute leukemias, Cancer Cell. 4 (2003) 99 – 110. [32] U. Fuchs, G. Rehkamp, O.A. Haas, R. Slany, M. Konig, S. Boiesen, R.M. Bohle, C. Damm-Welk, W.D. Ludwig, J. Harbott, A. Borkhardt, The human forming-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8756 – 8761. [33] V. Poupon, S. Polo, M. Vecchi, G. Martin, A. Dautry-Varsat, N. Cerf-Bensussan, P.P. Di Fiore, A. Benmerah, Differential nucleocytoplasmic trafficking between the related endocytic proteins Eps15 and Eps15R, J. Biol. Chem. 277 (2002) 8941 – 8948. [34] B. Rayet, Y. Fan, C. Gelinas, Mutations in the v-Rel transactivation domain indicate altered phosphorylation and identify a subset of NFkappaB-regulated cell death inhibitors important for v-Rel transforming activity, Mol. Cell. Biol. 23 (2003) 1520 – 1533. [35] L. Deng, C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart, Z.J. Chen, Activation of the I-kappa-B complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique poyubiquitin chain, Cell 103 (2000) 351 – 361. [36] S. Amit, Y. Ben-Neriah, NF-kappa B activation in cancer: a challenge for ubiquitination- and proteasome-based therapeutic approach, Semin. Cancer Biol. 13 (2003) 15 – 28.

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257 [37] D.K. Bonen, Y. Ogura, D.L. Nicolae, N. Inohara, L. Saab, T. Tanabe, F.F. Chen, S.J. Foster, R.H. Duerr, S.R. Brant, J.H. Cho, G. Nunez, Crohn’s disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan, Gastroenterology 124 (2003) 140 – 146. [38] K. Aoki, G. Meng, K. Suzuki, T. Takashi, Y. Kameoka, K. Nakahara, R. Ishida, M. Kasai, RP58 associates with condensed chromatin and mediates a sequence-specific transcriptional repression, J. Biol. Chem. 273 (1998) 26698 – 26704. [39] D. Perrotti, S. Bonatti, R. Trotta, R. Martinez, T. Skorski, P. Salomoni, E. Grassilli, R.V. Iozzo, D.R. Cooper, B. Calabretta, TLS/ FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis, EMBO J. 17 (1998) 4442 – 4455. [40] T.A. Kim, J. Lim, S. Ota, S. Raja, R. Rogers, B. Rivnay, H. Avraham, S. Avraham, NRP/B, a novel nuclear matrix protein, associates with p100(RB) and is involved in neuronal differentiation, J. Cell Biol. 141 (1998) 553 – 566. [41] J. Dakour, H. Li, D.W. Morrish, PL48: a novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation, Gene 185 (1997) 153 – 157. [42] F.F. Ferrara, F. Fazi, A. Bianchini, F. Padula, V. Gelmetti, S. Minucci, M. Mancini, P.G. Pelicci, F. Lo Coco, C. Nervi, Histone deacetylasetargeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia, Cancer Res. 61 (2001) 2 – 7. [43] T. Tani, Y. Sakai, Y. Shirai, M. Ohtake, K. Hatakeyama, Simultaneous development of Crohn’s disease and myelodysplastic syndrome progressing to acute myelocytic leukemia in a patient with a normal karyotype, J. Gastroenterol. 31 (1996) 599 – 602. [44] S.M. Schleicher, Oral isotretinoin and inflammatory bowel disease, J. Am. Acad. Dermatol. 13 (1985) 834 – 835. [45] F. Buss, J.P. Luzio, J. Kendrick-Jones, V.I. Myosin, An actin motor for membrane traffic and cell migration, Traffic 3 (2002) 851 – 858. [46] R. Rezzani, L. Rodella, R. Bianchi, Cyclosporine A affects the organization of cytoskeletal fibrillar proteins in rat thymus, Acta Histochem. 102 (2000) 57 – 67. [47] S. Takeda, A. Yamashita, K. Maeda, Y. Maeda, Structure of the core domain of human cardiac troponin in the Ca(2+) saturated form, Nature 424 (2003) 35 – 41. [48] D.A. De Angelis, P.E. Braun, 2V3V-Cyclic nucleotide 3V-phosphodiesterase binds to actin-based cytoskeletal elements in an isoprenylation-independent manner, J. Neurochem. 67 (1996) 943 – 951. [49] A. Habermann, T.A. Schroer, G. Griffiths, J.K. Burkhardt, Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles, J. Cell Sci. 114 (2001) 229 – 240. [50] F. Vuadens, D. Gasparini, C. Deon, J.C. Sanchez, D.F. Hochstrasser, P. Schneider, J.D. Tissot, Identification of specific proteins in different lymphocyte populations by proteomic tools, Proteomics 2 (2002) 105 – 111. [51] Z. Li, G.S. Davis, C. Mohr, M. Nain, D. Gemsa, Suppression of LPS-induced tumor necrosis factor-alpha gene expression by microtubule disrupting agents, Imunobiology 195 (1996) 640 – 654. [52] S.B. Snapper, F.S. Rosen, E. Mizoguchi, P. Cohen, W. Khan, C.H. Liu, T.L. Hagemann, S.P. Kwan, R. Ferrini, L. Davidson, A.K. Bhan, F.W. Alt, Wiskott – Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation, Immunity 9 (1998) 81 – 91. [53] P.P. Lemons, D. Chen, A.M. Bernstein, M.K. Bennett, S.W. Whiteheart, Regulated secretion in platelets: identification of elements of the platelet exocytosis machinery, Blood 90 (1997) 1490 – 1500. [54] P. Chardin, S. Paris, B. Antonny, S. Robineau, S. Beraud-Dufour, C.L. Jackson, M. Chabre, A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains, Nature 384 (1996) 481 – 484. [55] J.R. Morgan, K. Prasad, S. Jin, G.J. Augustine, E.M. Lafer, Eps15 homology domain-NPF motif interactions regulate clathrin coat as-

[56]

[57]

[58] [59]

[60]

[61]

[62] [63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

255

sembly during synaptic vesicle recycling, J. Biol. Chem. 278 (2003) 33583 – 33592. L. Jacobsen, P. Madsen, S.K. Moestrup, A.H. Lund, N. Tommerup, A. Nykjaer, L. Sottrup-Jensen, J. Gliemann, C.M. Petersen, Molecular characterization of a novel human hybrid-type receptor that binds the alpha(2)-macroglobulin receptor-associated protein, J. Biol. Chem. 271 (1996) 31379 – 31383. V. Florian, T. Schluter, R. Bohnensack, A new member of the sorting nexin family interacts with the C-terminus of P-selectin, Biochem. Biophys. Res. Commun. 281 (2001) 1045 – 1050. M. Forgac, Structure and properties of the vacuolar (H+)-ATPases, J. Biol. Chem. 274 (1999) 12951 – 12954. M. Bentham, S. Mazaleyrat, M. Harris, The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 downmodulation, J. Gen. Virol. 84 (2003) 2705 – 2713. T. Welte, S.S. Zhang, T. Wang, Z. Zhang, D.G. Hesslein, Z. Yin, A. Kano, Y. Iwamoto, E. Li, J.E. Craft, A.L. Bothwell, E. Fikrig, P.A. Koni, R.A. Flavell, X.Y. Fu, STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 1879 – 1884. P. Lovato, C. Brender, J. Agnholt, J. Kelsen, K. Kaltoft, A. Svejgaard, K.W. Eriksen, A. Woetmann, N. Odum, Constitutive STAT3 activation in intestinal T cells from patients with Crohn’s disease, J. Biol. Chem. 278 (2003) 16777 – 16781. O. Acuto, D. Cantrell, T cell activation and the cytoskeleton, Annu. Rev. Immunol. 18 (2000) 165 – 184. T. Zhang, J. Ma, X. Cao, Grb2 negatively regulates Stat3 activation in epidermal growth factor signaling, Biochem. J., (Elecronic publication ahead of print). C. Basbaum, D. Li, E. Gensch, M. Gallup, H. Lemjabbar, Mechanisms by which Gram-positive bacteria and tobacco smoke stimulate mucin induction through the epidermal growth factor receptor (EGFR), Novartis Found Symp. 248 (2002) 171 – 176. Y.-G. Ko, H. Park, T. Kim, J.-W. Lee, S.G. Park, W. Seol, J.E. Kim, W.-H. Lee, S.-H. Kim, J.-E. Park, S. Kim, A cofactor of tRNA synthetase, p43, is secreted to up-regulate proinflammatory genes, J. Biol. Chem. 276 (2001) 23028 – 23033. T.N. Basu, D.H. Gutmann, J.A. Fletcher, T.W. Glover, F.S. Collins, J. Downward, Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients, Nature 356 (1992) 713 – 715. S. Cedeno, D.F. Cirarelli, A.M. Blasini, M. Paris, F. Placeres, G. Alonso, M.A. Rodriguez, Defective activity of ERK-1 and ERK-2 mitogen-activated protein kinases in peripheral blood T lymphocytes from patients with systemic lupus erythematous: potential role of altered coupling of Ras guanine nucleotide exchange factor hSos to adapter protein Grb2 in lupus T cells, Clin. Immunol. 106 (1) (2003 Jan) 41 – 49. R.K. Lo, H. Cheung, Y.H. Wong, Constitutively active G(alpha)16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism, J. Biol. Chem., (Electronic publication ahead of print). R.E. Smith, V. Patel, S.D. Seatter, M.R. Deehan, M.H. Brown, G.P. Brooke, H.S. Goodridge, C.J. Howard, K.P. Rigley, W. Harnett, M.M. Harnett, A novel MyD-1 (SIRP-1alpha) signaling pathway that inhibits LPS-induced TNFalpha production by monocytes, Blood 102 (2003) 2532 – 2540. S. Latour, H. Tanaka, C. Demeure, V. Mateo, M. Rubio, E.J. Brown, C. Maliszewski, F.P. Lindberg, A. Oldenborg, A. Ulrich, G. Delespesse, M. Sarfati, Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation, J. Immunol. 167 (2001) 2547 – 2554. M. Zheng, P.J. McKeown-Longo, Regulation of HEF1 expression and phosphorylation by TGF-beta 1 and cell adhesion, J. Biol. Chem. 277 (2002) 39599 – 39608.

256

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257

[72] G.A. van Seventer, H.J. Salmen, S.F. Law, G.M. O’Neill, M.M. Mullen, A.M. Franz, S.B. Kanner, E.A. Golemis, J.M. van Seventer, Focal adhesion kinase regulates beta1 integrin-dependent T cell migration through an HEF1 effector pathway, Eur. J. Immunol. 31 (2001) 1417 – 1427. [73] A. Hirao, I. Hamaguchi, T. Suda, N. Yamaguchi, Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36anchored Lyn tyrosine kinase in thrombin-stimulated platelets, EMBO J. 16 (1997) 2342 – 2351. [74] L.R. Whyburn, K.E. Halcomb, C.M. Contreras, C.A. Lowell, O.N. Witte, A.B. Satterthwaite, Reduced dosage of Bruton’s tyrosine kinase uncouples B cell hyperresponsiveness from autoimmunity in lyn/ mice, J. Immunol. 171 (2003) 1850 – 1858. [75] P.E. Bickel, P.E. Scherer, J.E. Schnitzer, P. Oh, M.P. Lisanti, H.F. Lodish, Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins, J. Biol. Chem. 272 (1997) 13793 – 13802. [76] M. Simons, E.M. Kramer, C. Thiele, W. Stoffel, J. Trotter, Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains, J. Cell Biol. 151 (2000) 143 – 154. [77] S. Gaillard, M. Bartoli, F. Castets, A. Monneron, Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1, FEBS Lett. 508 (2001) 49 – 52. [78] G. Baillat, S. Gaillard, F. Castets, A. Monneron, Interactions of phocein with nucleoside-diphosphate kinase, Eps15, and Dynamin I, J. Biol. Chem. 277 (2002) 18961 – 18966. [79] S. Treves, G. Feriotto, L. Moccagatta, R. Gambari, F. Zorzato, Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane, J. Biol. Chem. 275 (2000) 39555 – 39568. [80] C.J. Speed, C.B. Neylon, P.J. Little, C.A. Mitchell, Underexpression of the 43kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responses following endothelin-1 stimulation, J. Cell Sci. 112 (1999) 669 – 679. [81] R.A. van Hogezand, R.F. Eichhorn, A. Choudry, R.A. Veenendaal, C.B. Lamers, Malignancies in inflammatory bowel disease: fact or fiction? Scand. J. Gastroenterol., Suppl. 236 (2002) 48 – 53. [82] J.E. Dinchuk, R.J. Focht, J.A. Kelley, N.L. Henderson, N.I. Zolotarjova, R. Wynn, N.T. Neff, J. Link, R.M. Huber, T.C. Burn, M.J. Rupar, M.R. Cunningham, B.H. Selling, J. Ma, A.A. Stern, G.F. Hollis, R.B. Stein, P.A. Friedman, Absence of post-translational aspartyl beta-hydroxylation of epidermal growth factor domains in mice leads to developmental defects and an increased incidence of intestinal neoplasia, J. Biol. Chem. 277 (2002) 12970 – 12977. [83] H. Shau, L.H. Butterfield, R. Chiu, A. Kim, Cloning and sequence analysis of candidate human natural killer-enhancing factor genes, Immunogenetics 40 (1994) 129 – 134. [84] J.B. de Haan, C. Bladier, P. Griffiths, M. Kelner, R.D. O’Shea, N.S. Cheung, R.T. Bronson, M.J. Silvestro, S. Wild, S.S. Zheng, P.M. Beart, P.J. Hertzog, I. Kola, Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide, J. Biol. Chem. 273 (1998) 22528 – 22536. [85] R. Kuner, P. Teismann, A. Trutzel, J. Naim, A. Richter, N. Schmidt, O. von Ahsen, A. Bach, B. Ferger, A. Schneider, TorsinA protects against oxidative stress in COS-1 and PC12 cells, Neurosci. Lett. 350 (2003) 153 – 156. [86] N. Jarrous, P.S. Eder, C. Guerrier-Takada, C. Hoog, S. Altman, Autoantigenic properties of some protein subunits of catalytically active complexes of human ribonuclease P, RNA 4 (1998) 407 – 417. [87] C.J. Simpson, L.A. Fothergill-Gilmore, Isolation and sequence of a cDNA encoding human platelet phosphofructokinsae, Biochem. Biophys. Res. Commun. 180 (1991) 197 – 203. [88] F.K. Lee, M.C. Cheung, S. Chung, The human sorbitol dehydroge-

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

nase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization, Genomics 21 (1994) 354 – 358. E. Goldmuntz, Z. Wang, B.A. Roe, M.L. Budarf, Cloning, genomic organization, and chromosomal localization of human citrate transport protein to the DiGeorge/velcardiofacial syndrome minimal critical region, Genomics 33 (1996) 271 – 276. J. Zhang, M. Kalkum, B.T. Chait, R.G. Roeder, The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2, Mol. Cell 9 (2002) 611 – 623. S.I. Matsuzawa, J.C. Reed, Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses, Mol. Cell. 7 (2001) 915 – 926. B.H. Ye, G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. de Waard, C. Leung, M. Nouri-Shirazi, A. Orazi, R.S.K. Chaganti, P. Rothman, A.M. Stall, P.-P. Pandolfi, R. Dalla-Favera, The Bcl-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation, Nat. Genet. 16 (1997) 161 – 170. K.P. Garrett, D. Haque, R.C. Conaway, J.W. Conaway, A human cDNA encoding the small subunit of RNA polymerase II transcription factor SIII, Gene 150 (1994) 413 – 414. W.J. Habets, P.T.G. Sillekens, M.H. Hoet, J.A. Schalken, A.J.M. Roebroek, J.A.M. Leunissen, W.J.M. van de Ven, W.J. Vendrooij, Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated Bdouble prime antigen, Proc. Nat. Acad. Sci. 84 (1987) 2421 – 2425. E. Kordeli, S. Lambert, V. Bennett, Ankyrin-G: a new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier, J. Biol. Chem. 270 (1995) 2353 – 2359. M. Simon, E. Girbal, M. Sebbag, V. Gomes-Daudrix, C. Vincent, G. Salama, G. Serre, The cytokeratin filament-aggregating protein filaggrin is the target of the so-called ‘‘antikeratin antibodies,’’ autoantibodies specific for rheumatoid arthritis, J. Clin. Invest. 92 (1993) 1387 – 1393. D. Pradhan, J. Morrow, The spectrin – ankyrin skeleton controls CD45 surface display and interleukin-2 production, Immunity 17 (2002) 303 – 315. T. Caballero, F. Nogueras, M.T. Medina, M.D. Caracuel, C. de Sola, F.J. Martinez-Salmeron, M. Rodrigo, R. Garcia del Moral, Intraepithelial and lamina propria leucocyte subsets in inflammatory bowel disease: an immunohistochemical study of colon and rectal biopsy specimens, J. Clin. Pathol. 48 (1995) 743 – 748. C.M. Hales, J.P. Vaerman, J.R. Goldenring, Rab11 family interacting protein 2 associates with Myosin Vb and regulates plasma membrane recycling, J. Biol. Chem. 277 (2002) 50415 – 50421. B. Panic, O. Perisic, D.B. Veprintsey, R.L. Williams, S. Munro, Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus, Mol. Cell 12 (2003) 863 – 874. M. Tarosounas, H.H. Heng, C.J. Ye, R.E. Pearlman, P.B. Moens, Identification of the mouse betaV-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9, Cytogenet. Cell Genet. 87 (1999) 201 – 204. D.G. Schwarz, C.T. Griffin, E.A. Schneider, D. Yee, T. Magnuson, Genetic analysis of sorting nexins 1 and 2 reveals a redundant and essential function in mice, Mol. Biol. Cell. 13 (2002) 3588 – 3600. N. Nishimura, H. Plutner, K. Hahn, W.E. Balch, The delta subunit of AP-3 is required for efficient transport of VSV-G from the transGolgi network to the cell surface, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 6755 – 6760. P. Kantheti, X. Oiao, M.E. Diaz, A.A. Peden, G.E. Meyer, S.L. Carskadon, D. Kapfhamer, D. Sufalko, M.S. Robinson, J.L. Noebels, M. Burmeister, Mutation in AP-3 delta in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles, Neuron 21 (1998) 111 – 122. R.A. Schinella, M.A. Greco, B.L. Cobert, et al, Hermansky – Pudlak syndrome with granulomatous colitis, Ann. Intern. Med. 92 (1) (1980 Jan) 20 – 23.

E.E. Mannick et al. / Clinical Immunology 112 (2004) 247–257 [106] H. Cao, W.E. Courchesne, C.C. Mastick, A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase, J. Biol. Chem. 277 (2002) 8771 – 8774. [107] T. Vang, H. Abrahamsen, S. Myklebust, V. Horeisi, K. Tasken, Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling, J. Biol. Chem. 16, 278 (2003) 17597 – 17600. [108] D. Agrawal, T. Chen, R. Irby, J. Quackenbush, A.F. Chambers, M. Szabo, A. Cantor, D. Coppola, T.J. Yeatman, Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling, J. Natl. Cancer Inst. 94 (2002) 513 – 521. [109] S. Ashkar, G.F. Weber, V. Panoutsakaopoulou, M.E. Sanchirico, M. Jansson, S. Zawaideh, S.R. Rittling, D.T. Denhardt, M.J. Glimchar, H. Cantor, Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity, Science 287 (2000) 860 – 864. [110] T. Tanaka-Kagawa, H. Jinno, T. Hasegawa, Y. Makino, Y. Seko, N. Hanioka, M. Ando, Functional characterization of two variant hu-

[111]

[112]

[113]

[114]

257

man GSTO 1-1s (Ala140Asp and Thr217Asn), Biochem. Biophys. Res. Commun. 301 (2003) 516 – 520. J. Bellingham, K. Gregory-Evans, M.F. Fox, C.Y. Gregory-Evans, Gene structure and tissue expression of human selenoprotein W, SEPW1, and identification of a retroprocessed pseudogene, SEPW1P, Biochim. Biophys. Acta 1627 (2003) 140 – 146. N.N. Danial, C.F. Gramm, L. Scorrano, C.-Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Gygi, S.P. Gygi, S.J. Korsmeyer, BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis, Nature 424 (2003) 952 – 956. N. Malik, V. Canfield, G. Sanchez-Watts, A.G. Watts, S. Scherer, B.G. Beatty, P. Gros, R. Levenson, Structural organization and chromosomal localization of the human Na,K-ATPase beta-3 subunit gene and pseudogene, Mamm. Genome 9 (1998) 136 – 143. W.T. Lowther, B.W. Matthews, Structure and function of the methionine aminopeptidases, Biochim. Biophys. Acta 1477 (2000) 157 – 167.