Intestinal Epithelial Cell Up-Regulation of LY6 Molecules during Colitis Results in Enhanced Chemokine Secretion

This information is current as Ken Flanagan, Zora Modrusan, Jennine Cornelius, Arvind of September 25, 2021. Chavali, Ian Kasman, Laszlo Komuves, Lian Mo and Lauri Diehl J Immunol 2008; 180:3874-3881; ; doi: 10.4049/jimmunol.180.6.3874

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Intestinal Epithelial Cell Up-Regulation of LY6 Molecules during Colitis Results in Enhanced Chemokine Secretion

Ken Flanagan,* Zora Modrusan,† Jennine Cornelius,* Arvind Chavali,* Ian Kasman,* Laszlo Komuves,* Lian Mo,* and Lauri Diehl1*

In the healthy colon, intestinal epithelial cells (IEC) form a physical barrier separating the myriad of gut Ags from the cells of the . Simultaneously, IEC use several mechanisms to actively maintain immunologic tolerance to nonpathogenic Ags, including commensal bacteria. However, during inflammatory bowel disease (IBD), the line of defense provided by IEC is breached, resulting in uncontrolled immune responses. As IEC are a principal mediator of immune responses in the gut, we were interested in discerning the expression pattern of IEC during development and progression of IBD. Laser capture micro- dissection and microarray analysis were combined to identify the LY6 superfamily as strongly up-regulated in inflamed IEC of the colon in two models of murine colitis. Surface expression of LY6A and LY6C on IEC is induced by several present Downloaded from within the colitic gut, including IL-22 and IFN-␥. Furthermore, cross-linking of LY6C results in production of a number of chemokines which are known to be involved in the immunopathogenesis of IBD. Increased chemokine production was cholesterol dependent, suggesting a role for lipid raft structures in the mechanism. As such, LY6 molecules represent novel targets to down-regulate chemokine expression in the colon and limit subsequent inflammation associated with IBD. The Journal of Im- munology, 2008, 180: 3874–3881. http://www.jimmunol.org/ ntestinal epithelial cells (IEC)2 form a barrier which is con- within the gut. It is believed that such immune recognition of non- tinuously exposed on the apical side to the numerous foreign pathogenic Ags underlies the pathology of IBD (7). I Ags within the lumen of the gut, and to cells of the immune IEC express a wide array of chemokines in response to an in- system on the basolateral side (1). The gut contains large numbers flammatory stimulus (8–10). Such chemokines serve to induce an of immune cells that stand poised to identify and respond to patho- influx of immune cells including neutrophils, dendritic cells, and T gens should they enter the body. The primary role of intestinal cells. For example, CXCL5, a chemokine molecule that attracts epithelium is to provide a physical barrier to separate foreign pro- neutrophils (11, 12) is up-regulated during inflammation, and leads teins from Ag presentation and subsequent immune responses. to increased neutrophil influx. Neutrophils play a number of roles by guest on September 25, 2021 Furthermore, mucosal B and T cells reside in the lamina propria, at the site of epithelial injury including phagocytosis, release of located adjacent to the intestinal epithelium (2). Coupled with the reactive oxygen species, and secretion of growth factors (13, 14). capability of IEC to process, transport, and present Ag (3, 4), IEC Neutrophils themselves also secrete a number of chemokines maintain a secondary role in maintaining immune tolerance, while which serve to increase the infiltration of other cells of the immune simultaneously initiating immune responses when potential patho- system including and T cells, amplifying the cascade gens are detected (5). of inflammation (15, 16). In the context of the non-IBD bowel, Inflammatory bowel disease (IBD) describes a group of related such actions serve to limit by initiating immune re- pathologies typified by acute and chronic intestinal inflammation sponses. However, when immune cell infiltration and activation in the absence of identifiable pathogen (6). By mechanisms which are divorced from pathogens, as appears to be the case in IBD, are not fully understood, patients with active IBD inappropriately such infiltration can be deleterious. trigger an inflammatory cascade of cytokines and chemokines In this study, we sought to discover novel molecules, expressed leading to initiation and propagation of immune responses to non- by IEC, that are dysregulated during colitis. Here, we describe the pathogenic Ags, such as commensal bacteria. Such inflammation expression of several members of the LY6 superfamily of genes, likely remains unresolved due to the multitude of Ags present which are not expressed on healthy IEC, but are strongly up-reg- ulated on the surface of IEC in models of murine colitis. The majority of LY6 family members are GPI-anchored cell surface *Department of Pathology and †Department of Molecular Biology, Genentech, South glycoproteins with broad distribution on cells of hematopoietic San Francisco, CA 94080 origin and more limited expression on nonhematopoietic cells. Received for publication August 2, 2007. Accepted for publication January 9, 2008. Though widely used as markers of differentiation of immune cells The costs of publication of this article were defrayed in part by the payment of page (18), the functions that the LY6 family possess have been difficult charges. This article must therefore be hereby marked advertisement in accordance to elucidate (19). Reports have shown that LY6 molecules are with 18 U.S.C. Section 1734 solely to indicate this fact. involved in a diverse array of functions including T cell activation 1 Address correspondence and reprint requests to Dr. Lauri Diehl, Genentech, 1 DNA Way, MS 72B, South San Francisco, CA 94080. E-mail address: Diehl.Lauri@ (20, 21), olfaction (22), and cellular adhesion (23). gene.com This report demonstrates that expression of LY6 family mem- 2 Abbreviations used in this paper: IEC, intestinal epithelial cell; IBD, inflammatory bers, particularly LY6A and LY6C, on the surface of IEC is reg- bowel disease; KLH, keyhole limpet hemocyanin; LCM, laser capture microscopy; ulated by inflammatory cytokines, including TNF-␣, IFN-␥, and siRNA, small interfering RNA. IL-22. We present evidence that cross-linking of LY6 family mem- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 bers on the surface of IEC results in increased production of many www.jimmunol.org The Journal of Immunology 3875 chemokines by a mechanism that appears to involve cholesterol, 488-conjugated streptavidin, mounted using Prolong Gold with 4Ј,6Ј-dia- suggesting a role for lipid raft reorganization in LY6-mediated midino-2-phenylindole (Invitrogen Life Technologies), and visualized by chemokine production. This data suggests that LY6 family mem- confocal microscopy. bers may be involved in the pathogenesis of IBD by establishing Cross-linking LY6 molecules and maintaining an unresolved chemokine gradient in the colon. For cross-linking using plate-bound Ab, 100 ␮l of the indicated concen- tration of the indicated Ab was added to a 96-well plate, or 2 ml were added Materials and Methods to a 60-mm2 dish and incubated overnight at 4°C. Plates were washed Reagents, cells, and mice extensively before cells were added. IFN-␥, TNF-␣, and IL-1␤ were obtained from PeproTech. IFN-␣ was ob- Silencing LY6C tained from Hycult Biotechnology. IL-22 was obtained from R&D Sys- tems. For cross-linking experiments, anti-keyhole limpet hemocyanin Individual small interfering RNA (siRNA) directed against murine LY6C (KLH) control Ab, anti-LY6A (clone E13-161.7 or D7) were obtained were obtained from Dharmacon. siRNA was transfected into YAMC cells from BD Pharmingen. Anti-LY6C (clone HK1.4) was obtained from using Lipofectamine 2000 (Invitrogen Life Technologies) and standard Southern Biotechnology Associates. protocols. Seventy-two hours after transfection, cells were collected to de- Chronic CD45RBhigh transfer colitis was induced as described previ- termine knockdown efficiency. One siRNA was chosen for cross-linking ously in SCID mice on a BALB/c background (24). IL-10Ϫ/Ϫ mice (25) on experiments based on superior knockdown efficiency (95% inhibition by a 129 background, which develop spontaneous colitis, were sacrificed be- quantitative RT-PCR). tween 11 and 13 wk of age. Colons were snap-frozen in OCT until used in CXCL5 secretion experiments as described. Proximal colon, middle colon, distal colon, and rectum were scored using a scale of 0–5 (0, normal bowel; 5, severe dis- Supernatants were collected at the indicated time point from stimulated ease). Scores were summed to achieve a total colitis severity score for each cells and CXCL5 concentrations were determined by ELISA using a com- Downloaded from animal. mercially available kit from R&D Systems and manufacturers’ protocols. The young adult mouse colonocyte (YAMC) cell line (provided by R. The level of detection was at least 15 pg/ml CXCL5. Whitehead, Vanderbilt University Medical Center, Nashville, TN) was de- rived from the Immortomouse, a transgenic animal containing a tempera- Cholesterol depletion ture-sensitive T Ag under the control of an IFN-␥-dependent promoter, as previously described (26). YAMC cells proliferate under permissive con- YAMC cells were cultured for 72 h in serum-free medium at 37°C in the ␥ presence of 4 ␮M lovastatin and 250 ␮M mevalonate (Sigma-Aldrich). ditions of 32°C in the presence of 5 U/ml IFN- (PeproTech), but no longer http://www.jimmunol.org/ proliferate upon removal of IFN-␥ at 37°C (nonpermissive conditions). Cells were plated and maintained in lovastatin and mevalonate throughout YAMC cells were cultured in RPMI 1640 containing 5% FBS, 2 mM the experiment. ␥ L-glutamine, penicillin/streptomycin, 5 U/ml IFN- , and N-2 supplement Statistics (Invitrogen Life Technologies). Cells were cultured under nonpermissive indicates ء) conditions for at least 24 h before experiments, and for the duration of The Student t test was used for comparison between groups experimentation. CMT93 cells were obtained from American Type Culture p Ͻ 0.05). Collection cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin. Results Laser capture microscopy (LCM) and RNA purification Expression of LY6 family members is strongly up-regulated on

the surface of colitic IEC by guest on September 25, 2021 Ten- to 12-␮m sections were applied to LCM membrane slides (Molecular Machines). Slides were subjected to an abbreviated H&E stain (total time Studies have indicated that patterns of IEC are of about 5 min) before crypt epithelial cells were histologically identified significantly altered in mouse models of colitis, as well as human and dissected using an MMI Cellcut microscope. RNA was purified from IBD (29–31). In this study, we evaluated gene expression patterns the dissected cells using the Arcturus Picopure RNA purification kit and the manufacturer’s protocols (Arcturus) and quantified by Nanodrop in IEC of healthy and colitic mice to illuminate novel genes and (Nanodrop Technologies). pathways altered in IBD. We were interested in identifying genes involved in the immu- Microarray hybridization and data analysis nopathology of IBD, and evaluated IEC from the CD45RBhigh T Ϫ Ϫ Total RNA sample was converted to double-stranded labeled cDNA using cell transfer colitis model as well as the IL-10 / model, both of a Low RNA Input Fluorescent Linear Amplification kit. Approximately which are believed to result from Th1 dysregulation and share 500,000 counts of Cy-dye-labeled cRNA was fragmented and hybridized to many features of human Crohn’s disease (32, 33). Laser capture the Agilent whole mouse array as described in Agilent’s In Situ Hybridization kit Plus. LCM samples were labeled with Cy5 dye and hy- microdissection was used to isolate crypt IEC from the colons of bridized against Cy3 dye-labeled universal mouse reference. Following healthy and colitic mice in these two models of murine IBD. RNA hybridization, the arrays were washed, dried with acetonitrile, and scanned was extracted from these samples and analyzed by microarray. The on Agilent’s DNA microarray scanner. gene expression profile of IEC of colitic mice in the transfer colitis Data were analyzed using Rosetta Resolver software (Rosetta Biosoft- Ͼ ware). Briefly, healthy and colitic samples were grouped separately and model identified 1770 probes with 2-fold expression changes Ϫ/Ϫ probes that passed two-tailed ANOVA ( p Ͻ 0.05) were selected. These compared with control mice, while the IL-10 model identified probes were analyzed further for probes that demonstrated a 2-fold or 1140 probes. Overlapping in both models, there were 540 probes greater change in colitic samples vs healthy samples. with Ͼ2-fold changes in expression, corresponding to ϳ400 Real-time quantitative RT-PCR unique genes (data not shown). Members of the LY6 family of molecules were overrepresented RT-PCR was performed on extracted RNA using TaqMan Gold RT-PCR in number as well as degree of up-regulation in both the transfer kit and reagents (Applied Biosystems). All samples were run with gene- Ϫ/Ϫ specific primers using 5Ј-FAM and 3Ј-TAMRA-labeled internal probes. colitis model and the IL-10 model (Fig. 1). These results were Analysis was performed compared with housekeeping gene, SPF31, spe- confirmed by real-time quantitative RT-PCR of pooled and ampli- cific primers by the 2Ϫ⌬⌬Ct method (where Ct is the cycle threshold) as fied IEC RNA in the transfer colitis model (data not shown). Ex- described (27). Primers and probes were either designed using Primer3 pression of the LY6 family members was unique to the disease software (28) or obtained commercially (Applied Biosystems). state, as no healthy mice expressed appreciable levels of any of Immunofluorescent staining these LY6 family members. Frozen tissues were cut into 5-␮m sections and stained with biotinylated Although expression of these LY6 molecules on the surface of anti-LY6C (Southern Biotechnology Associates) or anti-LY6A at 2.5 cells of hematopoietic origin is known, expression on IEC has not ng/ml (R&D Systems). Slides were washed and labeled with Alexa Fluor been previously described (34, 35). As expression of LY6A and 3876 LY6C EXPRESSION IN COLITIC MICE RAISES CHEMOKINE PRODUCTION

Transcription of LY6 genes is stimulated by inflammatory cytokines LY6 expression on T cells is induced and enhanced by both type I and type II IFNs (36). Furthermore, expression of a number of cytokines is elevated in the colon during active colitis (37). To determine whether cytokines present during colitis affect transcription of LY6 family members in IEC, we treated YAMC cells, a conditionally immortalized murine IEC line, with IL-1␤, FIGURE 1. LY6 family members are up-regulated in IEC in murine Ϫ Ϫ IFN-␣, TNF-␣, IFN-␥, or the combination of TNF-␣ and IFN-␥ models of colitis. IEC in both the IL-10 / (left) and CD45RBhigh transfer colitis model (right) were isolated by LCM and RNA was purified. Mi- and analyzed the transcription of all identified murine LY6 genes croarray analysis was performed and analyzed as described in Materials by real-time quantitative RT-PCR (Table I). Although many of the and Methods. Numbers to the right represent the average mean of the fold LY6 family members were not detected in either the presence or change compared with the universal standard RNA of colitic mice over absence of inflammatory cytokines (see the Table I legend), we healthy mice. Numbers below the heat map indicate the inflammation score detected a strong up-regulation in the transcription of LY6A, of the individual mouse. LY6C, and LY6F in response to the majority of the cytokines tested, as well as more moderate up-regulation of LY6E, LY6H, and LYPD1 in response to some cytokines tested. However, IFN-␥

was by far the most potent in inducing LY6 up-regula- Downloaded from LY6C is detectable on many nonepithelial cells present within the tion. Furthermore, TNF-␣ enhanced the effects of IFN-␥ on the colon, such as T cells and granulocytes, it remained possible that expression of LY6A, LY6F, LY6E, and LYPD1. the microarray was detecting LY6 molecule RNA due to an in- To examine the surface expression of LY6 family members in crease in the influx of contaminating immune cells. To rule out this response to cytokines, YAMC cells were exposed to the above possibility, we performed immunofluorescent staining and confo- cytokines and analyzed by flow cytometry for expression of LY6A cal microscopy for both LY6A and LY6C on healthy and colitic and LY6C, for which commercial Abs are available. Unlike http://www.jimmunol.org/ colons. Although levels of LY6A and LY6C were minimal or ab- healthy IEC in situ, high levels of LY6A were expressed on sent on the surface of healthy IEC (Fig. 2, A and C, respectively), YAMC cells even in the absence of added cytokines (Fig. 3B, expression of both LY6A and LY6C was detectable on the surface medium). Expression of LY6C (Fig. 3A, medium) was consider- of IEC throughout the colons of colitic mice (Fig. 2, B and D, ably lower than expression of LY6A. IL-1␤ and TNF-␣ induced respectively). There was no evidence of polarization of either slight increases in the surface expression of both LY6A and LY6C, LY6A or LY6C, and staining was present on both the apical and in agreement with RNA expression. A more moderate increase in basolateral membranes, making LY6 molecules potentially acces- expression was noted when IFN-␣ was added to the cells, while sible to ligands on either surface. There was no obvious polariza- IFN-␥ induced dramatic increases in surface expression of both tion of either LY6A (Fig. 2E) or LY6C (Fig. 2F). by guest on September 25, 2021 LY6A and LY6C; surface expression closely mirrored RNA expression. Th2 cytokines, such as IL-4, IL-10, or IL-13 had no effect on surface expression of either LY6A or LY6C (data not shown). Induction of both LY6A (Fig. 3D) and LY6C (Fig. 3C)by IFN-␥ was dose dependent. Doses as low as 6.25 U/ml IFN-␥ resulted in detectable increases in both LY6 molecules by flow cytometry. Furthermore, the increase in both LY6A (Fig. 3F) and LY6C (Fig. 3E) surface expression became evident between 2 and 4 h after IFN-␥ treatment, and steadily increased for at least 24 h after IFN-␥ treatment. This data indicates that relatively low con- centrations of IFN-␥ are sufficient to increase surface expression of LY6 molecules within hours. There is evidence that IL-22, which is secreted primarily from activated T cells, functions through the IL-22R complex, present on IEC to promote cytokine production and an inflammatory phe- notype (30). Furthermore, IL-22 is involved in the immunopatho- genesis of Crohn’s disease. To examine whether IL-22 affects LY6 molecule expression on murine IEC, YAMC cells were cultured in the presence of IL-22 and analyzed for expression of LY6C (Fig. 3G) and LY6A (Fig. 3H). Both LY6 molecules were substantially increased in the presence of IL-22 at comparable levels to the induction seen after treatment with IFN-␥. To ensure that the up-regulation of LY6 molecules was not spe- cific to the YAMC cell line, we looked at RNA levels of LY6A and FIGURE 2. Surface expression of LY6 molecules are up-regulated on Ϫ Ϫ Ϫ Ϫ LY6C in the murine colonic epithelial tumor cell line CMT93. IEC in the IL-10 / model of colitis. Wild-type (A) or IL-10 / mice (B) were stained for surface expression of LY6A (red, with DAPI counter- Levels of both LY6A and LY6C were up-regulated upon treatment Ϫ Ϫ ␥ stain). Similarly, wild-type (C) or IL-10 / mice (D) were stained for with IFN- (Fig. 3I). This data supports the data obtained by real- surface expression of LY6C. There was no obvious polarization of either time quantitative RT-PCR in confirming that IEC up-regulate LY6 LY6A (E) or LY6C (F) in IL-10Ϫ/Ϫ mice at higher magnifications. family members in response to inflammatory cytokines. The Journal of Immunology 3877

Table I. IEC up-regulate LY6 family members in response to inflammatory cytokinesa

Refseq Media IL-1␤ IFN-␣ TNF-␣ IFN-␥ IFN-␥ ϩ TNF-␣

Ly6A NM_010738 1.0 Ϯ 0.0 1.8 Ϯ 0.3 2.8* Ϯ 0.5 2.2* Ϯ 0.4 33.1* Ϯ 5.2 65.4*† Ϯ 4.3 Ly6C NM_010741 1.0 Ϯ 0.0 1.6* Ϯ 0.3 2.4* Ϯ 0.2 1.2* Ϯ 0.0 65.6* Ϯ 8.2 63.6* Ϯ 4.1 Ly6D NM_010742 1.0 Ϯ 0.1 2.7* Ϯ 0.5 1.5* Ϯ 0.2 2.1* Ϯ 0.1 1.0 Ϯ 0.0 0.9 Ϯ 0.2 Ly6E NM_008529 1.0 Ϯ 0.0 1.4* Ϯ 0.2 2.1* Ϯ 0.1 1.5* Ϯ 0.2 1.9* Ϯ 0.2 2.9*† Ϯ 0.2 Ly6F NM_008530 1.0 Ϯ 0.0 2.5* Ϯ 0.3 7.1* Ϯ 1.3 0.6* Ϯ 0.1 108.2* Ϯ 15.2 169.7*† Ϯ 10.6 Ly6H NM_011837 1.0 Ϯ 0.2 1.0 Ϯ 0.0 1.2 Ϯ 0.4 1.1 Ϯ 0.1 3.7* Ϯ 1.2 1.4† Ϯ 0.3 Lypd1 NM_145100 1.0 Ϯ 0.1 1.5* Ϯ 0.5 1.3* Ϯ 0.0 2.1* Ϯ 0.2 1.3* Ϯ 0.1 2.9*† Ϯ 0.3 Lypd2 NM_026671 1.0 Ϯ 0.3 0.1* Ϯ 0.0 0.4* Ϯ 0.1 ND 0.1* Ϯ 0.1 ND Ly6g5c NM_148947 1.1 Ϯ 0.4 1.3 Ϯ 0.3 0.9 Ϯ 0.1 0.8 Ϯ 0.3 1.3 Ϯ 0.4 1.1 Ϯ 0.3 Ly6g6c NM_023463 1.0 Ϯ 0.1 0.7 Ϯ 0.3 0.6 Ϯ 0.4 0.7 Ϯ 0.3 0.6* Ϯ 0.2 0.3*† Ϯ 0.2 Slurp2/Lynx1 NM_011838 1.1 Ϯ 0.3 0.7 Ϯ 0.5 0.7 Ϯ 0.4 0.4 Ϯ 0.5 1.5 Ϯ 0.5 0.4* Ϯ 0.2

a RNA levels of the indicated LY6 family member in IEC were determined by real-time quantitative RT-PCR after 15 h of treatment with the indicated cytokine. Number represents the fold change (determined by 2Ϫ⌬⌬Ct method) vs the untreated, media control. The following LY6 family members were tested, but not detected in samples, regardless of treatment: LY6K, Lypd3, Lypd4, Lypd5, LY6g5b, Ly6g6d, Ly6g6e, and Slurp1. ND: No detected Ct. None detected: Ly6K, Lypd4, Ly6g6e, Lypd5, Lypd3, Ly6g5b, .p Ͻ 0.05 vs media control; †, p Ͻ 0.05 vs IFN-␥-treated cells ,ء .and Slurp1

Stimulation of LY6C results in increased secretion of Downloaded from chemokines Functions for LY6 molecules have not been fully elucidated. Fur- thermore, expression of many of these family members was pre- viously believed to be limited to cells of hematopoietic origin. Therefore, we wished to determine what, if any, role LY6 expres-

sion on IEC might have on epithelial cell biology and in the im- http://www.jimmunol.org/ munopathology of colitis. To determine whether any gene tran- scription pathways are affected by LY6C cross-linking, YAMC cells, either pretreated with IFN-␥ to up-regulate LY6 molecule expression, or untreated, were cultured on plates coated with anti- KLH control Ab, or anti-LY6C. RNA from these cells was ob- tained 8 h later, and analyzed by microarray. Although only ϳ40 by guest on September 25, 2021

FIGURE 3. Surface expression of LY6A and LY6C are up-regulated in response to inflammatory cytokines, particularly IFN-␥. YAMC cells were FIGURE 4. Cross-linking of LY6C affects chemokine expression. A, treated with the indicated cytokine (100 ng/ml, except for IFN-␥ at 100 Culture dishes (60 mm2) were coated with the indicated Ab at 10 ␮g/ml. U/ml) for 15 h and stained for surface expression of LY6C (A) and LY6A YAMC cells, either pretreated for 15 h with 100 U/ml IFN-␥, or left un- (B). YAMC cells were cultured for 15 h in the presence of increasing doses treated, as indicated (Ϫ or ϩ), were added for 24 h. RNA was extracted and of IFN-␥ and analyzed by flow cytometry for expression of LY6C (C) and analyzed for chemokine expression by real-time quantitative RT-PCR. LY6A (D). IFN-␥-stimulated YAMC cells were collected at various time Data represents mean fold change vs RNA from nonpretreated, anti-KLH points, as indicated, and analyzed by flow cytometry for expression of cross-linked cells as determined by the 2Ϫ⌬⌬Ct method. CCL3, XCL1, and LY6C (E) and LY6A (F). IL-22 up-regulated expression of both LY6C (G) CCL20 were also analyzed, but no expression was detected in samples, and LY6A (H). RNA levels of LY6C and LY6A were similarly up-regu- regardless of treatment. B, Levels of both CXCL5 and CXCL2 in response lated in the murine IEC line, CMT93, in response to treatment with IFN-␥ to LY6C cross-linking were diminished when LY6C levels were knocked (I). down with siRNA. 3878 LY6C EXPRESSION IN COLITIC MICE RAISES CHEMOKINE PRODUCTION Downloaded from

FIGURE 5. Cross-linking LY6C, but not LY6A, induces secretion of chemokines. YAMC cells were preincubated or not, as indicated, with 100 U/ml IFN-␥ for 15 h (as indicated) and cultured on plates coated with 10 ␮g/ml anti-LY6A (f), anti-LY6C (z), or anti-KLH control (Ⅺ). RNA was isolated at 24, 48, and 72 h and analyzed for expression of CXCL5 (A)or

CCL7 (B). Data indicate mean Ϯ SD of the fold change (as determined by http://www.jimmunol.org/ Ϫ⌬⌬ FIGURE 6. LY6-mediated up-regulation of chemokines is decreased by 2 Ct method) compared with untreated, isotype cross-linked cells. C, cholesterol depletion. A, Disruption of lipid raft integrity by cholesterol Supernatants were collected at 48 h in cells cross-linked, as above, with 1, depletion results in an inhibition of LY6C-mediated chemokine produc- 5, or 10 ␮g/ml (as indicated) of Ab and CXCL5 secretion into the super- tion. Cholesterol-depleted or nondepleted YAMC cells (as indicated below natant was determined by ELISA. the graph) were incubated with plate-bound anti-KLH or anti-LY6C as indicated for 15 h. RNA was collected and expression levels of CXCL2, CXCL5, and CCL7 were determined. Surface levels of LY6A (B) and genes were affected by LY6C cross-linking, chemokine genes LY6C (C) were decreased in response to cholesterol depletion. were widely up-regulated upon LY6C cross-linking (data not shown). by guest on September 25, 2021 To confirm this, we analyzed LY6C and LY6A cross-linked (Fig. 4B). Secretion of CXCL5 was markedly inhibited by knock- YAMC cell RNA by quantitative RT-PCR for expression of ing down LY6C as well (data not shown). CCL2, CCL4, CCL5, CCL7, CCL8, CCL25, CXCL1, CXCL2, To analyze the kinetics of chemokine up-regulation induced by CXCL5, CXCL10, CXCL12, and CX3CL1, which are chemokines LY6C stimulation, 96-well plates were coated with anti-KLH Ab that have been implicated in colitis (Fig. 4A) (38–40). The assay or either anti-LY6A or anti-LY6C mAbs. YAMC cells, either pre- was performed under nonpermissive growth conditions (37°C in treated or not with IFN-␥, were added for 24, 48, or 72 h. At the the absence of IFN-␥) to rule out the possibility of increased pro- indicated time point, RNA was collected for quantitative RT-PCR liferation of IEC in response to IFN-␥ stimulation. analysis and supernatants were collected for ELISA. Cells pretreated with IFN-␥ showed up-regulation of many of Within 24 h, a spike in transcription of both CXCL5 (Fig. 5A) these chemokine genes (medium, anti-KLH group vs IFN-␥, anti- and CCL7 (Fig. 5B) was detected on cells with cross-linked LY6C, KLH group). With the exception of a moderate up-regulation of but not LY6A. Increased expression of both CXCL5 and CCL7 CCL8 and a down-regulation of CXCL1, anti-LY6A-stimulated diminished over time but was still detectable after 72 h in culture. YAMC cells showed similar gene expression patterns as anti- Though IFN-␥ was not required to enhance chemokine transcrip- KLH-stimulated YAMC cells. However, YAMC cells stimulated tion, IFN-␥ acted synergistically with LY6C stimulation in induc- with anti-LY6C showed increased expression of all chemokines ing transcription of both CXCL5 and CCL7 at early time points. analyzed except for CCL25, which remained essentially un- In parallel with the gene expression, supernatants of LY6C, but changed, and CXCL12, which was moderately down-regulated in not LY6A, cross-linked cells contained significantly higher con- response to LY6C stimulation. Although the increased gene ex- centrations of CXCL5 at 48 h (Fig. 5C). The effect was dose de- pression of chemokines induced by LY6C cross-linking was not pendent, and detectable with as little as 1 ␮g/ml coated anti-LY6C. dependent upon IFN-␥, cells pretreated with IFN-␥ did show in- Like transcription, secretion of CXCL5 was enhanced when cells creased expression of chemokines vs cells that had not been pre- were pretreated with IFN-␥, but IFN-␥ was not required for the treated with IFN-␥. effect. Increased secretion of CXCL5 was noted at both the 24 and To ensure that LY6C was involved in the observed up-regula- 72 h time points as well (data not shown). These results indicate tion of chemokines, we used siRNA to knockdown LY6C. LY6C that cross-linking of LY6C, but not LY6A, on the surface of IEC transcript was inhibited by 95% in the absence of IFN-␥ and results in increased secretion of chemokines. ϳ90% in the presence of IFN-␥ by real-time quantitative RT-PCR which corresponded to significantly lower levels of LY6C on the LY6 up-regulation of chemokine secretion is dependent upon surface of the YAMC cells (data not shown). Cells with decreased cholesterol biosynthesis levels of LY6C on the surface showed a diminished response to As GPI-anchored , LY6 family members do not possess a LY6C cross-linking with regard to transcription of chemokines unique intracellular domain associated with traditional outside-in The Journal of Immunology 3879

fluenced by cholesterol biosynthesis, allows for the expression of LY6 molecules on the surface, and is potentially involved in the LY6C-mediated induction of chemokines.

Cross-linking LY6C results in increased surface expression of LY6 molecules It has been reported that cross-linking LY6C on the surface of T cells results in shedding of LY6C (23). However, our microarray data indicated that when LY6C was cross-linked on YAMC cells, there was significant up-regulation of transcription of LY6A, LY6C, and LY6D (data not shown). To confirm the up-regulation of LY6 family members in response to LY6C cross-linking, we analyzed surface expression of LY6A and LY6C after cross-link- ing YAMC cells with LY6C. FIGURE 7. Cross-linking of LY6C, but not LY6A, induces up-regula- Unlike T cells, when LY6C was cross-linked on the surface of tion of surface expression of LY6A and LY6C. YAMC cells were incu- IEC, no shedding of either LY6A or LY6C occurred (Fig. 7, B and bated for 24 h on plates coated with anti-KLH control, anti-LY6A, or A, respectively). To the contrary, in the absence of IFN-␥, surface

A Downloaded from anti-LY6C and analyzed by flow cytometry for expression of LY6C ( )or expression levels of both LY6A and LY6C were increased on IEC LY6A (B). Cells were pretreated for 12 h with 100 U/ml IFN-␥ and sim- with cross-linked LY6C, but not LY6A. When IEC were preincu- ilarly plated on Ab-coated plates and analyzed for expression of LY6C (C) ␥ or LY6A (D). bated with IFN- , much of this effect was abolished (Fig. 7C); however, a slight up-regulation of LY6A was still detected (Fig. 7D). This data suggests a positive feedback loop whereby stimu- signaling. Rather, they are present within lipid raft microdomains lation through LY6C on IEC results in increased surface expres- (41). It has been suggested that cross-linking of GPI-anchored pro- sion of LY6 molecules. http://www.jimmunol.org/ teins family members on the surface of cells results in redistribu- tion of other cell surface molecules as well as reorganization of IEC in vivo show a similar chemokine gene expression to lipid raft structures, possibly explaining how LY6 molecules can LY6C-stimulated cells affect signal transduction and downstream cellular functions (42). Cholesterol is required to maintain lipid raft integrity (43), and The above data establish a model whereby IEC stimulated through depletion of cholesterol is often used to inhibit lipid raft biosyn- LY6C significantly up-regulate expression of chemokine genes. thesis in vitro (44). When YAMC cells were pretreated with lo- Analyzing the microarray data from laser capture microdissected

vastatin and mevalonate to inhibit cholesterol biosynthesis before IEC in murine models of colitis, we looked at the expression of the by guest on September 25, 2021 LY6C cross-linking, there was a significant decrease in the up- same 12 chemokine genes in healthy and colitic mice in the two regulation of CXCL2, CXCL5, and CCL7, indicating that when murine models of colitis to determine whether the chemokines lipid rafts are depleted, the LY6C-mediated up-regulation of che- stimulated by LY6C cross-linking in vitro correlate with the che- mokine genes is diminished (Fig. 6A). Cholesterol depletion af- mokines secreted by IEC in vivo (Fig. 8). Though the expression fected chemokine production in control anti-KLH stimulated pattern is not identical with the up-regulation of chemokines re- groups, irrespective of LY6C stimulation; however, the response sulting from LY6C stimulation, we see that expression of CXCL5, was minimal and not in a consistent direction. To confirm that which was the most highly up-regulated chemokine gene in in cholesterol depletion was not globally affecting cell viability, we vitro studies, was also the highest up-regulated chemokine in mu- measured cell death, by 7-aminoactinomycin D exclusion, and de- rine models of colitis. We saw significant up-regulation in expres- termined that cholesterol depletion did not significantly affect the sion of CXCL1, CXCL10, CCL5, and CCL7 in both models of viability of the YAMC cells (92% viability vs 86% in the choles- colitis. In addition, we saw up-regulation of CCL4 and CCL8 in terol-depleted cells, data not shown). the transfer colitis model or the IL-10Ϫ/Ϫ model, respectively. Surface expression of both LY6A (Fig. 6B) and LY6C (Fig. It is interesting to note that CXCL12, the only chemokine that 6C) were significantly lower in cholesterol-depleted YAMC was down-regulated as a result of LY6C stimulation in vitro, was cells, suggesting that plasma membrane cholesterol levels and also the only one of these chemokines down-regulated during co- lipid raft integrity affect the levels of LY6 expression on the litis in vivo suggesting that LY6C cross-linking may be involved surface of cells. This data suggests that lipid raft integrity, in- in the in vivo up-regulation of chemokines during colitis.

FIGURE 8. IEC in colitis possess a similar che- mokine gene expression pattern. IEC in both the IL- 10Ϫ/Ϫ (left) and CD45RBhigh transfer colitis model (right) were isolated by LCM and RNA was puri- fied. Microarray analysis was performed and ana- lyzed as described in Materials and Methods. Num- bers represent the mean of the fold change compared with the universal standard RNA of colitic mice over healthy mice. Numbers below the heat map indicate the inflammation score of the individual mouse. 3880 LY6C EXPRESSION IN COLITIC MICE RAISES CHEMOKINE PRODUCTION

Discussion IEC. As IBD is viewed as an unresolved inflammatory response Expression of LY6 molecules has been previously studied in the (56), it is possible that once LY6 molecules are up-regulated on the context of hematopoietic cells (45). Though largely used as mark- surface of IEC, likely as a result of IFN-␥ as well as other cyto- ers of differentiation and activation of such cells, roles for LY6 kines present in the colitic gut, stimulation of these molecules re- molecules in diverse processes such as T cell activation and ad- sults in even further LY6 expression thereby establishing a posi- hesion have been described (21, 23, 46). In this report, we describe tive feedback loop that would maintain LY6 expression in the expression of LY6 molecules on the surface of IEC, and further absence of pathogens. characterize that expression is unique to IEC in the context of It has previously been reported that not only is chemokine ex- inflammation. LY6 molecule expression was not noted on healthy pression up-regulated during active colitis, but that chemokines are epithelial cells, but RNA and surface expression levels of LY6A involved in disease initiation and progression (38). In our own and LY6C were high on IEC of colitic mice, and nearly universally microarray studies presented here, it is clear that IEC greatly up- expressed on epithelial cells throughout the colon. As molecules regulate expression of a number of chemokine genes, including both specific to the diseased state, and ubiquitously expressed dur- CCL7 and CXCL5. ing disease, LY6 molecules represent an interesting candidate for Our data indicate that there is a possibility that lipid raft integ- a role in the pathogenesis of colitis. rity is involved in LY6C-mediated signal transduction in IEC. This We determined functional significance of LY6 expression in implies that disruption of lipid rafts might serve to attenuate down- IEC using a previously described conditionally immortalized mu- stream affects of LY6C stimulation both by down-regulating LY6C expression and disrupting the structural components of rine epithelial cell line, YAMC. Unlike IEC in vivo which lacked LY6C signaling. Recently, it has been determined that cholesterol Downloaded from expression of LY6A or LY6C by immunofluorescent staining, un- depletion of IEC with statins inhibits proinflammatory gene ex- stimulated YAMC cells were strongly positive for LY6A, and ex- pression through NF-␬B modulation (57). Furthermore, statins pressed lower levels of LY6C. However, upon stimulation with a have been effective therapeutics in murine models of colitis (17). number of cytokines present within the colon during colitis, in- The mechanism linking lipid raft motility and NF-␬B blockade cluding IL-1␤, TNF-␣, IFN-␣, and, in particular, IL-22 and IFN-␥, remain undetermined, but our data suggest that activation through expression levels of both LY6 molecules were greatly enhanced.

LY6C could be one hypothesis to explain the mechanism of action. http://www.jimmunol.org/ As such, we considered YAMC cells an appropriate in vitro model In this study, we identify LY6 molecules as a potential upstream to analyze functional significance for LY6 expression. switch in the expression of chemokine genes. Cross-linking of the It should be noted that the conditionally immortalized nature of LY6C receptor with mAbs resulted in dramatic up-regulation of the YAMC cells comes from MHC class II promoter-driven ex- nearly all chemokines analyzed, including CXCL5. We further pression of the SV40 large T Ag; low levels (2.5–5 U/ml) of IFN-␥ confirmed that CXCL5 secretion is greatly enhanced in LY6C are used to drive proliferation of these cells (26, 47). YAMC cells cross-linked IEC. are often used as an in vitro model for cytokine treatments of It is interesting that even though both LY6A and LY6C are murine IEC (48, 49). The SV40 large T Ag that these cells con- anchored to the cell surface by a GPI moiety, and despite higher tained is temperature sensitive, and nonfunctional at 37°C. We levels of expression of LY6A than LY6C on the surface of IEC, by guest on September 25, 2021 ␥ therefore performed all experiments involving IFN- treatment un- the downstream effects on chemokine secretion are seen with der these nonpermissive conditions. In addition, YAMC cells were LY6C cross-linking and not consistently with LY6A cross-linking. ␥ serum starved (and IFN- starved) at 37°C for 24 h before exper- We hope to determine whether chemokine secretion occurs fol- iments. Under such conditions, effects indicating residual T Ag lowing stimulation of other LY6 family members, particularly expression, such as proliferation of cells, were not noted. We be- LY6D, LY6E, LY6F, or LY6I, all of which were up-regulated in ␥ lieve that effects of IFN- treatment are due to inherent effects of colitic IEC, or whether LY6C is unique in this regard. IFN-␥ rather than effects stemming from driving expression of the T Ag. Furthermore, the up-regulation of LY6 family members was Acknowledgments detected in a second murine cell line, CMT93, lending further We thank Roli Khattri and Ken Refino for help with obtaining mouse support to the hypothesis that this effect is broadly applicable to tissues. We also thank Andres Paler Martinez for his help with flow cy- IEC. Furthermore, IFN-␥ was not unique among cytokines for in- tometry, Alex Abbas for his advice regarding analysis of microarray data, ducing LY6 molecules as modest up-regulation of LY6 expression and Laura Sanders for assistance with figures. was noted after treatment with TNF-␣, IL-1␤, and IL-22. The up- regulation of LY6 molecules on IEC in response to IL-22 is in- Disclosures teresting in light of recent data demonstrating a potential role for All authors are current or former employees of Genentech, Inc. No cur- rently marketed Genentech products are mentioned in this manuscript but IL-22 in Crohn’s disease (50). it is possible the research could contribute to future commercial products or Though homology between mouse and human LY6 molecules is impact other drug development processes. often complicated, there is evidence to suggest that the up-regu- lation of LY6 molecules is not restricted to mice. Previous studies References in rats have suggested up-regulation of LY6 molecules in the small 1. Tlaskalova-Hogenova, H., R. Stepankova, T. Hudcovic, L. Tuckova, intestine in colitis models, and it has been suggested that such B. Cukrowska, R. Lodinova-Zadnikova, H. Kozakova, P. Rossmann, J. Bartova, D. Sokol, et al. 2004. Commensal bacteria (normal microflora), mucosal immu- expression is involved in inflammation, cell/cell interactions, as nity and chronic inflammatory and autoimmune diseases. Immunol. Lett. 93: well as signaling within the rat IEC (51). 97–108. 2. Campbell, N., X. Y. Yio, L. P. So, Y. Li, and L. Mayer. 1999. The intestinal Ligands for LY6 molecules, including LY6A, have been de- epithelial cell: processing and presentation of antigen to the mucosal immune scribed on the lymphoid cells; however, the identification of such system. Immunol. Rev. 172: 315–324. ligands is controversial (52–55). Furthermore, ligands have not 3. Mayer, L. 2000. Epithelial cell antigen presentation. Curr. Opin. Gastroenterol. 16: 531–535. been wholly identified. To study LY6 ligand binding, cross-linking 4. Hershberg, R. M., and L. F. Mayer. 2000. Antigen processing and presentation by studies using mAbs have been performed (23). intestinal epithelial cells–polarity and complexity. Immunol. Today 21: 123–128. 5. Neutra, M. R., N. J. Mantis, and J. P. Kraehenbuhl. 2001. Collaboration of ep- It is interesting to note that LY6C stimulation was sufficient to ithelial cells with organized mucosal lymphoid tissues. Nat. Immunol. 2: up-regulate expression of both LY6A and LY6C on the surface of 1004–1009. The Journal of Immunology 3881

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