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The Journal of Immunology, 2008, 180, 3874-3881
Copyright © 2008 by The American Association of Immunologists, Inc.
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Intestinal Epithelial Cell Up-Regulation of LY6 Molecules during Colitis Results in Enhanced Chemokine Secretion

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

* Department of Pathology and {dagger} Department of Molecular Biology, Genentech, South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the healthy colon, intestinal epithelial cells (IEC) form a physical barrier separating the myriad of gut Ags from the cells of the immune system. 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 gene expression pattern of IEC during development and progression of IBD. Laser capture microdissection and microarray analysis were combined to identify the LY6 superfamily as strongly up-regulated genes in inflamed IEC of the colon in two models of murine colitis. Surface expression of LY6A and LY6C on IEC is induced by several cytokines present within the colitic gut, including IL-22 and IFN-{gamma}. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intestinal epithelial cells (IEC)2 form a barrier which is continuously exposed on the apical side to the numerous foreign Ags within the lumen of the gut, and to cells of the immune system on the basolateral side (1). The gut contains large numbers of immune cells that stand poised to identify and respond to pathogens should they enter the body. The primary role of intestinal epithelium is to provide a physical barrier to separate foreign proteins from Ag presentation and subsequent immune responses.

Furthermore, mucosal B and T cells reside in the lamina propria, located adjacent to the intestinal epithelium (2). Coupled with the capability of IEC to process, transport, and present Ag (3, 4), IEC maintain a secondary role in maintaining immune tolerance, while simultaneously initiating immune responses when potential pathogens are detected (5).

Inflammatory bowel disease (IBD) describes a group of related pathologies typified by acute and chronic intestinal inflammation in the absence of identifiable pathogen (6). By mechanisms which are not fully understood, patients with active IBD inappropriately trigger an inflammatory cascade of cytokines and chemokines leading to initiation and propagation of immune responses to nonpathogenic Ags, such as commensal bacteria. Such inflammation likely remains unresolved due to the multitude of Ags present within the gut. It is believed that such immune recognition of nonpathogenic Ags underlies the pathology of IBD (7).

IEC express a wide array of chemokines in response to an inflammatory stimulus (8, 9, 10). Such chemokines serve to induce an influx of immune cells including neutrophils, dendritic cells, and T cells. For example, CXCL5, a chemokine molecule that attracts neutrophils (11, 12) is up-regulated during inflammation, and leads to increased neutrophil influx. Neutrophils play a number of roles at the site of epithelial injury including phagocytosis, release of reactive oxygen species, and secretion of growth factors (13, 14). Neutrophils themselves also secrete a number of chemokines which serve to increase the infiltration of other cells of the immune system including macrophages and T cells, amplifying the cascade of inflammation (15, 16). In the context of the non-IBD bowel, such actions serve to limit infection by initiating immune responses. However, when immune cell infiltration and activation are divorced from pathogens, as appears to be the case in IBD, such infiltration can be deleterious.

In this study, we sought to discover novel molecules, expressed by IEC, that are dysregulated during colitis. Here, we describe the expression of several members of the LY6 superfamily of genes, which are not expressed on healthy IEC, but are strongly up-regulated on the surface of IEC in models of murine colitis. The majority of LY6 family members are GPI-anchored cell surface glycoproteins with broad distribution on cells of hematopoietic origin and more limited expression on nonhematopoietic cells. Though widely used as markers of differentiation of immune cells (18), the functions that the LY6 family possess have been difficult to elucidate (19). Reports have shown that LY6 molecules are involved in a diverse array of functions including T cell activation (20, 21), olfaction (22), and cellular adhesion (23).

This report demonstrates that expression of LY6 family members, particularly LY6A and LY6C, on the surface of IEC is regulated by inflammatory cytokines, including TNF-{alpha}, IFN-{gamma}, and IL-22. We present evidence that cross-linking of LY6 family members on the surface of IEC results in increased production of many chemokines by a mechanism that appears to involve cholesterol, suggesting a role for lipid raft reorganization in LY6-mediated chemokine production. This data suggests that LY6 family members may be involved in the pathogenesis of IBD by establishing and maintaining an unresolved chemokine gradient in the colon.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents, cells, and mice

IFN-{gamma}, TNF-{alpha}, and IL-1β were obtained from PeproTech. IFN-{alpha} was obtained from Hycult Biotechnology. IL-22 was obtained from R&D Systems. For cross-linking experiments, anti-keyhole limpet hemocyanin (KLH) control Ab, anti-LY6A (clone E13-161.7 or D7) were obtained from BD Pharmingen. Anti-LY6C (clone HK1.4) was obtained from Southern Biotechnology Associates.

Chronic CD45RBhigh transfer colitis was induced as described previously in SCID mice on a BALB/c background (24). IL-10–/– mice (25) on a 129 background, which develop spontaneous colitis, were sacrificed between 11 and 13 wk of age. Colons were snap-frozen in OCT until used in experiments as described. Proximal colon, middle colon, distal colon, and rectum were scored using a scale of 0–5 (0, normal bowel; 5, severe disease). Scores were summed to achieve a total colitis severity score for each animal.

The young adult mouse colonocyte (YAMC) cell line (provided by R. Whitehead, Vanderbilt University Medical Center, Nashville, TN) was derived from the Immortomouse, a transgenic animal containing a temperature-sensitive T Ag under the control of an IFN-{gamma}-dependent promoter, as previously described (26). YAMC cells proliferate under permissive conditions of 32°C in the presence of 5 U/ml IFN-{gamma} (PeproTech), but no longer proliferate upon removal of IFN-{gamma} at 37°C (nonpermissive conditions).

YAMC cells were cultured in RPMI 1640 containing 5% FBS, 2 mM L-glutamine, penicillin/streptomycin, 5 U/ml IFN-{gamma}, and N-2 supplement (Invitrogen Life Technologies). Cells were cultured under nonpermissive conditions for at least 24 h before experiments, and for the duration of experimentation. CMT93 cells were obtained from American Type Culture Collection cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin.

Laser capture microscopy (LCM) and RNA purification

Ten- to 12-µm sections were applied to LCM membrane slides (Molecular Machines). Slides were subjected to an abbreviated H&E stain (total time of about 5 min) before crypt epithelial cells were histologically identified and dissected using an MMI Cellcut microscope. RNA was purified from the dissected cells using the Arcturus Picopure RNA purification kit and the manufacturer’s protocols (Arcturus) and quantified by Nanodrop (Nanodrop Technologies).

Microarray hybridization and data analysis

Total RNA sample was converted to double-stranded labeled cDNA using a Low RNA Input Fluorescent Linear Amplification kit. Approximately 500,000 counts of Cy-dye-labeled cRNA was fragmented and hybridized to the Agilent whole mouse genome array as described in Agilent’s In Situ Hybridization kit Plus. LCM samples were labeled with Cy5 dye and hybridized against Cy3 dye-labeled universal mouse reference. Following hybridization, the arrays were washed, dried with acetonitrile, and scanned on Agilent’s DNA microarray scanner.

Data were analyzed using Rosetta Resolver software (Rosetta Biosoftware). Briefly, healthy and colitic samples were grouped separately and probes that passed two-tailed ANOVA (p < 0.05) were selected. These probes were analyzed further for probes that demonstrated a 2-fold or greater change in colitic samples vs healthy samples.

Real-time quantitative RT-PCR

RT-PCR was performed on extracted RNA using TaqMan Gold RT-PCR kit and reagents (Applied Biosystems). All samples were run with gene-specific primers using 5'-FAM and 3'-TAMRA-labeled internal probes. Analysis was performed compared with housekeeping gene, SPF31, specific primers by the 2–{Delta}{Delta}Ct method (where Ct is the cycle threshold) as described (27). Primers and probes were either designed using Primer3 software (28) or obtained commercially (Applied Biosystems).

Immunofluorescent staining

Frozen tissues were cut into 5-µm sections and stained with biotinylated anti-LY6C (Southern Biotechnology Associates) or anti-LY6A at 2.5 ng/ml (R&D Systems). Slides were washed and labeled with Alexa Fluor 488-conjugated streptavidin, mounted using Prolong Gold with 4',6'-diamidino-2-phenylindole (Invitrogen Life Technologies), and visualized by confocal microscopy.

Cross-linking LY6 molecules

For cross-linking using plate-bound Ab, 100 µl of the indicated concentration of the indicated Ab was added to a 96-well plate, or 2 ml were added to a 60-mm2 dish and incubated overnight at 4°C. Plates were washed extensively before cells were added.

Silencing LY6C

Individual small interfering RNA (siRNA) directed against murine LY6C were obtained from Dharmacon. siRNA was transfected into YAMC cells using Lipofectamine 2000 (Invitrogen Life Technologies) and standard protocols. Seventy-two hours after transfection, cells were collected to determine knockdown efficiency. One siRNA was chosen for cross-linking experiments based on superior knockdown efficiency (95% inhibition by quantitative RT-PCR).

CXCL5 secretion

Supernatants were collected at the indicated time point from stimulated cells and CXCL5 concentrations were determined by ELISA using a commercially available kit from R&D Systems and manufacturers’ protocols. The level of detection was at least 15 pg/ml CXCL5.

Cholesterol depletion

YAMC cells were cultured for 72 h in serum-free medium at 37°C in the presence of 4 µM lovastatin and 250 µM mevalonate (Sigma-Aldrich). Cells were plated and maintained in lovastatin and mevalonate throughout the experiment.

Statistics

The Student t test was used for comparison between groups (* indicates p < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of LY6 family members is strongly up-regulated on the surface of colitic IEC

Studies have indicated that gene expression patterns of IEC are significantly altered in mouse models of colitis, as well as human IBD (29, 30, 31). In this study, we evaluated gene expression patterns in IEC of healthy and colitic mice to illuminate novel genes and pathways altered in IBD.

We were interested in identifying genes involved in the immunopathology of IBD, and evaluated IEC from the CD45RBhigh T cell transfer colitis model as well as the IL-10–/– model, both of which are believed to result from Th1 dysregulation and share many features of human Crohn’s disease (32, 33). Laser capture microdissection was used to isolate crypt IEC from the colons of healthy and colitic mice in these two models of murine IBD. RNA was extracted from these samples and analyzed by microarray. The gene expression profile of IEC of colitic mice in the transfer colitis model identified 1770 probes with >2-fold expression changes compared with control mice, while the IL-10–/– model identified 1140 probes. Overlapping in both models, there were 540 probes with >2-fold changes in expression, corresponding to ~400 unique genes (data not shown).

Members of the LY6 family of molecules were overrepresented in number as well as degree of up-regulation in both the transfer colitis model and the IL-10–/– model (Fig. 1). These results were confirmed by real-time quantitative RT-PCR of pooled and amplified IEC RNA in the transfer colitis model (data not shown). Expression of the LY6 family members was unique to the disease state, as no healthy mice expressed appreciable levels of any of these LY6 family members.


Figure 1
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  		FIGURE 1. LY6 family members are up-regulated in IEC in murine models of colitis. IEC in both the IL-10–/– (left) and CD45RBhigh transfer colitis model (right) were isolated by LCM and RNA was purified. Microarray analysis was performed and analyzed as described in Materials and Methods. Numbers to the right represent the average 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.

 
Although expression of these LY6 molecules on the surface of cells of hematopoietic origin is known, expression on IEC has not been previously described (34, 35). As expression of LY6A and LY6C is detectable on many nonepithelial cells present within the colon, such as T cells and granulocytes, it remained possible that the microarray was detecting LY6 molecule RNA due to an increase in the influx of contaminating immune cells. To rule out this possibility, we performed immunofluorescent staining and confocal microscopy for both LY6A and LY6C on healthy and colitic colons. Although levels of LY6A and LY6C were minimal or absent on the surface of healthy IEC (Fig. 2, A and C, respectively), expression of both LY6A and LY6C was detectable on the surface of IEC throughout the colons of colitic mice (Fig. 2, B and D, respectively). There was no evidence of polarization of either LY6A or LY6C, and staining was present on both the apical and basolateral membranes, making LY6 molecules potentially accessible to ligands on either surface. There was no obvious polarization of either LY6A (Fig. 2E) or LY6C (Fig. 2F).


Figure 2
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  		FIGURE 2. Surface expression of LY6 molecules are up-regulated on 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 counterstain). Similarly, wild-type (C) or IL-10–/– mice (D) were stained for surface expression of LY6C. There was no obvious polarization of either LY6A (E) or LY6C (F) in IL-10–/– mice at higher magnifications.

 
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-1β, IFN-{alpha}, TNF-{alpha}, IFN-{gamma}, or the combination of TNF-{alpha} and IFN-{gamma} and analyzed the transcription of all identified murine LY6 genes by real-time quantitative RT-PCR (Table I). Although many of the LY6 family members were not detected in either the presence or absence of inflammatory cytokines (see the Table I legend), we detected a strong up-regulation in the transcription of LY6A, 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-{gamma} was by far the most potent cytokine in inducing LY6 up-regulation. Furthermore, TNF-{alpha} enhanced the effects of IFN-{gamma} on the expression of LY6A, LY6F, LY6E, and LYPD1.


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

 
To examine the surface expression of LY6 family members in response to cytokines, YAMC cells were exposed to the above cytokines and analyzed by flow cytometry for expression of LY6A and LY6C, for which commercial Abs are available. Unlike healthy IEC in situ, high levels of LY6A were expressed on YAMC cells even in the absence of added cytokines (Fig. 3B, medium). Expression of LY6C (Fig. 3A, medium) was considerably lower than expression of LY6A. IL-1β and TNF-{alpha} induced slight increases in the surface expression of both LY6A and LY6C, in agreement with RNA expression. A more moderate increase in expression was noted when IFN-{alpha} was added to the cells, while IFN-{gamma} induced dramatic increases in surface expression of both LY6A and LY6C; surface protein 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).


Figure 3
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  		FIGURE 3. Surface expression of LY6A and LY6C are up-regulated in response to inflammatory cytokines, particularly IFN-{gamma}. YAMC cells were treated with the indicated cytokine (100 ng/ml, except for IFN-{gamma} at 100 U/ml) for 15 h and stained for surface expression of LY6C (A) and LY6A (B). YAMC cells were cultured for 15 h in the presence of increasing doses of IFN-{gamma} and analyzed by flow cytometry for expression of LY6C (C) and LY6A (D). IFN-{gamma}-stimulated YAMC cells were collected at various time points, as indicated, and analyzed by flow cytometry for expression of LY6C (E) and LY6A (F). IL-22 up-regulated expression of both LY6C (G) and LY6A (H). RNA levels of LY6C and LY6A were similarly up-regulated in the murine IEC line, CMT93, in response to treatment with IFN-{gamma} (I).

 
Induction of both LY6A (Fig. 3D) and LY6C (Fig. 3C) by IFN-{gamma} was dose dependent. Doses as low as 6.25 U/ml IFN-{gamma} 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-{gamma} treatment, and steadily increased for at least 24 h after IFN-{gamma} treatment. This data indicates that relatively low concentrations of IFN-{gamma} 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 phenotype (30). Furthermore, IL-22 is involved in the immunopathogenesis 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-{gamma}.

To ensure that the up-regulation of LY6 molecules was not specific to the YAMC cell line, we looked at RNA levels of LY6A and LY6C in the murine colonic epithelial tumor cell line CMT93. Levels of both LY6A and LY6C were up-regulated upon treatment with IFN-{gamma} (Fig. 3I). This data supports the data obtained by real-time quantitative RT-PCR in confirming that IEC up-regulate LY6 family members in response to inflammatory cytokines.

Stimulation of LY6C results in increased secretion of chemokines

Functions for LY6 molecules have not been fully elucidated. Furthermore, expression of many of these family members was previously believed to be limited to cells of hematopoietic origin. Therefore, we wished to determine what, if any, role LY6 expression on IEC might have on epithelial cell biology and in the immunopathology of colitis. To determine whether any gene transcription pathways are affected by LY6C cross-linking, YAMC cells, either pretreated with IFN-{gamma} 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 obtained 8 h later, and analyzed by microarray. Although only ~40 genes were affected by LY6C cross-linking, chemokine genes were widely up-regulated upon LY6C cross-linking (data not shown).

To confirm this, we analyzed LY6C and LY6A cross-linked YAMC cell RNA by quantitative RT-PCR for expression of CCL2, CCL4, CCL5, CCL7, CCL8, CCL25, CXCL1, CXCL2, CXCL5, CXCL10, CXCL12, and CX3CL1, which are chemokines that have been implicated in colitis (Fig. 4A) (38, 39, 40). The assay was performed under nonpermissive growth conditions (37°C in the absence of IFN-{gamma}) to rule out the possibility of increased proliferation of IEC in response to IFN-{gamma} stimulation.


Figure 4
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  		FIGURE 4. Cross-linking of LY6C affects chemokine expression. A, Culture dishes (60 mm2) were coated with the indicated Ab at 10 µg/ml. YAMC cells, either pretreated for 15 h with 100 U/ml IFN-{gamma}, or left untreated, as indicated (– or +), were added for 24 h. RNA was extracted and analyzed for chemokine expression by real-time quantitative RT-PCR. Data represents mean fold change vs RNA from nonpretreated, anti-KLH cross-linked cells as determined by the 2–{Delta}{Delta}Ct method. CCL3, XCL1, and CCL20 were also analyzed, but no expression was detected in samples, regardless of treatment. B, Levels of both CXCL5 and CXCL2 in response to LY6C cross-linking were diminished when LY6C levels were knocked down with siRNA.

 
Cells pretreated with IFN-{gamma} showed up-regulation of many of these chemokine genes (medium, anti-KLH group vs IFN-{gamma}, anti-KLH group). With the exception of a moderate up-regulation of CCL8 and a down-regulation of CXCL1, anti-LY6A-stimulated YAMC cells showed similar gene expression patterns as anti-KLH-stimulated YAMC cells. However, YAMC cells stimulated with anti-LY6C showed increased expression of all chemokines analyzed except for CCL25, which remained essentially unchanged, and CXCL12, which was moderately down-regulated in response to LY6C stimulation. Although the increased gene expression of chemokines induced by LY6C cross-linking was not dependent upon IFN-{gamma}, cells pretreated with IFN-{gamma} did show increased expression of chemokines vs cells that had not been pretreated with IFN-{gamma}.

To ensure that LY6C was involved in the observed up-regulation of chemokines, we used siRNA to knockdown LY6C. LY6C transcript was inhibited by 95% in the absence of IFN-{gamma} and ~90% in the presence of IFN-{gamma} by real-time quantitative RT-PCR which corresponded to significantly lower levels of LY6C on the surface of the YAMC cells (data not shown). Cells with decreased levels of LY6C on the surface showed a diminished response to LY6C cross-linking with regard to transcription of chemokines (Fig. 4B). Secretion of CXCL5 was markedly inhibited by knocking down LY6C as well (data not shown).

To analyze the kinetics of chemokine up-regulation induced by LY6C stimulation, 96-well plates were coated with anti-KLH Ab or either anti-LY6A or anti-LY6C mAbs. YAMC cells, either pretreated or not with IFN-{gamma}, were added for 24, 48, or 72 h. At the indicated time point, RNA was collected for quantitative RT-PCR analysis and supernatants were collected for ELISA.

Within 24 h, a spike in transcription of both CXCL5 (Fig. 5A) and CCL7 (Fig. 5B) was detected on cells with cross-linked LY6C, but not LY6A. Increased expression of both CXCL5 and CCL7 diminished over time but was still detectable after 72 h in culture. Though IFN-{gamma} was not required to enhance chemokine transcription, IFN-{gamma} acted synergistically with LY6C stimulation in inducing transcription of both CXCL5 and CCL7 at early time points.


Figure 5
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  		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-{gamma} for 15 h (as indicated) and cultured on plates coated with 10 µg/ml anti-LY6A ({blacksquare}), anti-LY6C (Figure 5), or anti-KLH control ({square}). RNA was isolated at 24, 48, and 72 h and analyzed for expression of CXCL5 (A) or CCL7 (B). Data indicate mean ± SD of the fold change (as determined by 2–{Delta}{Delta}Ct method) compared with untreated, isotype cross-linked cells. C, Supernatants were collected at 48 h in cells cross-linked, as above, with 1, 5, or 10 µg/ml (as indicated) of Ab and CXCL5 secretion into the supernatant was determined by ELISA.

 
In parallel with the gene expression, supernatants of LY6C, but not LY6A, cross-linked cells contained significantly higher concentrations of CXCL5 at 48 h (Fig. 5C). The effect was dose dependent, and detectable with as little as 1 µg/ml coated anti-LY6C. Like transcription, secretion of CXCL5 was enhanced when cells were pretreated with IFN-{gamma}, but IFN-{gamma} was not required for the effect. Increased secretion of CXCL5 was noted at both the 24 and 72 h time points as well (data not shown). These results indicate that cross-linking of LY6C, but not LY6A, on the surface of IEC results in increased secretion of chemokines.

LY6 up-regulation of chemokine secretion is dependent upon cholesterol biosynthesis

As GPI-anchored proteins, LY6 family members do not possess a unique intracellular domain associated with traditional outside-in signaling. Rather, they are present within lipid raft microdomains (41). It has been suggested that cross-linking of GPI-anchored proteins family members on the surface of cells results in redistribution of other cell surface molecules as well as reorganization of lipid raft structures, possibly explaining how LY6 molecules can affect signal transduction and downstream cellular functions (42).

Cholesterol is required to maintain lipid raft integrity (43), and depletion of cholesterol is often used to inhibit lipid raft biosynthesis in vitro (44). When YAMC cells were pretreated with lovastatin and mevalonate to inhibit cholesterol biosynthesis before LY6C cross-linking, there was a significant decrease in the up-regulation of CXCL2, CXCL5, and CCL7, indicating that when lipid rafts are depleted, the LY6C-mediated up-regulation of chemokine genes is diminished (Fig. 6A). Cholesterol depletion affected chemokine production in control anti-KLH stimulated groups, irrespective of LY6C stimulation; however, the response was minimal and not in a consistent direction. To confirm that cholesterol depletion was not globally affecting cell viability, we measured cell death, by 7-aminoactinomycin D exclusion, and determined that cholesterol depletion did not significantly affect the viability of the YAMC cells (92% viability vs 86% in the cholesterol-depleted cells, data not shown).


Figure 6
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  		FIGURE 6. LY6-mediated up-regulation of chemokines is decreased by cholesterol depletion. A, Disruption of lipid raft integrity by cholesterol depletion results in an inhibition of LY6C-mediated chemokine production. Cholesterol-depleted or nondepleted YAMC cells (as indicated below 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 LY6C (C) were decreased in response to cholesterol depletion.

 
Surface expression of both LY6A (Fig. 6B) and LY6C (Fig. 6C) were significantly lower in cholesterol-depleted YAMC cells, suggesting that plasma membrane cholesterol levels and lipid raft integrity affect the levels of LY6 expression on the surface of cells. This data suggests that lipid raft integrity, influenced 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-linking YAMC cells with LY6C.

Unlike T cells, when LY6C was cross-linked on the surface of IEC, no shedding of either LY6A or LY6C occurred (Fig. 7, B and A, respectively). To the contrary, in the absence of IFN-{gamma}, surface expression levels of both LY6A and LY6C were increased on IEC with cross-linked LY6C, but not LY6A. When IEC were preincubated with IFN-{gamma}, 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 stimulation through LY6C on IEC results in increased surface expression of LY6 molecules.


Figure 7
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  		FIGURE 7. Cross-linking of LY6C, but not LY6A, induces up-regulation of surface expression of LY6A and LY6C. YAMC cells were incubated for 24 h on plates coated with anti-KLH control, anti-LY6A, or anti-LY6C and analyzed by flow cytometry for expression of LY6C (A) or LY6A (B). Cells were pretreated for 12 h with 100 U/ml IFN-{gamma} and similarly plated on Ab-coated plates and analyzed for expression of LY6C (C) or LY6A (D).

 
IEC in vivo show a similar chemokine gene expression to LY6C-stimulated cells

The above data establish a model whereby IEC stimulated through LY6C significantly up-regulate expression of chemokine genes. Analyzing the microarray data from laser capture microdissected IEC in murine models of colitis, we looked at the expression of the same 12 chemokine genes in healthy and colitic mice in the two murine models of colitis to determine whether the chemokines stimulated by LY6C cross-linking in vitro correlate with the chemokines secreted by IEC in vivo (Fig. 8). Though the expression pattern is not identical with the up-regulation of chemokines resulting from LY6C stimulation, we see that expression of CXCL5, which was the most highly up-regulated chemokine gene in in vitro studies, was also the highest up-regulated chemokine in murine models of colitis. We saw significant up-regulation in expression of CXCL1, CXCL10, CCL5, and CCL7 in both models of colitis. In addition, we saw up-regulation of CCL4 and CCL8 in the transfer colitis model or the IL-10–/– model, respectively.


Figure 8
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  		FIGURE 8. IEC in colitis possess a similar chemokine gene expression pattern. IEC in both the IL-10–/– (left) and CD45RBhigh transfer colitis model (right) were isolated by LCM and RNA was purified. Microarray analysis was performed and analyzed as described in Materials and Methods. Numbers 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.

 
It is interesting to note that CXCL12, the only chemokine that was down-regulated as a result of LY6C stimulation in vitro, was also the only one of these chemokines down-regulated during colitis in vivo suggesting that LY6C cross-linking may be involved in the in vivo up-regulation of chemokines during colitis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Expression of LY6 molecules has been previously studied in the context of hematopoietic cells (45). Though largely used as markers of differentiation and activation of such cells, roles for LY6 molecules in diverse processes such as T cell activation and adhesion have been described (21, 23, 46). In this report, we describe expression of LY6 molecules on the surface of IEC, and further characterize that expression is unique to IEC in the context of inflammation. LY6 molecule expression was not noted on healthy epithelial cells, but RNA and surface expression levels of LY6A and LY6C were high on IEC of colitic mice, and nearly universally expressed on epithelial cells throughout the colon. As molecules both specific to the diseased state, and ubiquitously expressed during disease, LY6 molecules represent an interesting candidate for a role in the pathogenesis of colitis.

We determined functional significance of LY6 expression in IEC using a previously described conditionally immortalized murine epithelial cell line, YAMC. Unlike IEC in vivo which lacked expression of LY6A or LY6C by immunofluorescent staining, unstimulated YAMC cells were strongly positive for LY6A, and expressed lower levels of LY6C. However, upon stimulation with a number of cytokines present within the colon during colitis, including IL-1β, TNF-{alpha}, IFN-{alpha}, and, in particular, IL-22 and IFN-{gamma}, expression levels of both LY6 molecules were greatly enhanced. As such, we considered YAMC cells an appropriate in vitro model to analyze functional significance for LY6 expression.

It should be noted that the conditionally immortalized nature of the YAMC cells comes from MHC class II promoter-driven expression of the SV40 large T Ag; low levels (2.5–5 U/ml) of IFN-{gamma} are used to drive proliferation of these cells (26, 47). YAMC cells are often used as an in vitro model for cytokine treatments of murine IEC (48, 49). The SV40 large T Ag that these cells contained is temperature sensitive, and nonfunctional at 37°C. We therefore performed all experiments involving IFN-{gamma} treatment under these nonpermissive conditions. In addition, YAMC cells were serum starved (and IFN-{gamma} starved) at 37°C for 24 h before experiments. Under such conditions, effects indicating residual T Ag expression, such as proliferation of cells, were not noted. We believe that effects of IFN-{gamma} treatment are due to inherent effects of IFN-{gamma} rather than effects stemming from driving expression of the T Ag. Furthermore, the up-regulation of LY6 family members was detected in a second murine cell line, CMT93, lending further support to the hypothesis that this effect is broadly applicable to IEC. Furthermore, IFN-{gamma} was not unique among cytokines for inducing LY6 molecules as modest up-regulation of LY6 expression was noted after treatment with TNF-{alpha}, IL-1β, and IL-22. The up-regulation of LY6 molecules on IEC in response to IL-22 is interesting in light of recent data demonstrating a potential role for IL-22 in Crohn’s disease (50).

Though homology between mouse and human LY6 molecules is often complicated, there is evidence to suggest that the up-regulation of LY6 molecules is not restricted to mice. Previous studies in rats have suggested up-regulation of LY6 molecules in the small intestine in colitis models, and it has been suggested that such expression is involved in inflammation, cell/cell interactions, as well as signaling within the rat IEC (51).

Ligands for LY6 molecules, including LY6A, have been described on the lymphoid cells; however, the identification of such ligands is controversial (52, 53, 54, 55). Furthermore, ligands have not been wholly identified. To study LY6 ligand binding, cross-linking studies using mAbs have been performed (23).

It is interesting to note that LY6C stimulation was sufficient to up-regulate expression of both LY6A and LY6C on the surface of IEC. As IBD is viewed as an unresolved inflammatory response (56), it is possible that once LY6 molecules are up-regulated on the surface of IEC, likely as a result of IFN-{gamma} as well as other cytokines present in the colitic gut, stimulation of these molecules results in even further LY6 expression thereby establishing a positive feedback loop that would maintain LY6 expression in the absence of pathogens.

It has previously been reported that not only is chemokine expression up-regulated during active colitis, but that chemokines are involved in disease initiation and progression (38). In our own microarray studies presented here, it is clear that IEC greatly up-regulate expression of a number of chemokine genes, including CCL7 and CXCL5.

Our data indicate that there is a possibility that lipid raft integrity is involved in LY6C-mediated signal transduction in IEC. This implies that disruption of lipid rafts might serve to attenuate downstream affects of LY6C stimulation both by down-regulating LY6C expression and disrupting the structural components of LY6C signaling. Recently, it has been determined that cholesterol depletion of IEC with statins inhibits proinflammatory gene expression through NF-{kappa}B modulation (57). Furthermore, statins have been effective therapeutics in murine models of colitis (17). The mechanism linking lipid raft motility and NF-{kappa}B blockade remain undetermined, but our data suggest that activation through LY6C could be one hypothesis to explain the mechanism of action.

In this study, we identify LY6 molecules as a potential upstream switch in the expression of chemokine genes. Cross-linking of the LY6C receptor with mAbs resulted in dramatic up-regulation of nearly all chemokines analyzed, including CXCL5. We further confirmed that CXCL5 secretion is greatly enhanced in LY6C cross-linked IEC.

It is interesting that even though both LY6A and LY6C are anchored to the cell surface by a GPI moiety, and despite higher levels of expression of LY6A than LY6C on the surface of IEC, the downstream effects on chemokine secretion are seen with LY6C cross-linking and not consistently with LY6A cross-linking. We hope to determine whether chemokine secretion occurs following stimulation of other LY6 family members, particularly LY6D, LY6E, LY6F, or LY6I, all of which were up-regulated in colitic IEC, or whether LY6C is unique in this regard.


   Acknowledgments
 
We thank Roli Khattri and Ken Refino for help with obtaining mouse tissues. We also thank Andres Paler Martinez for his help with flow cytometry, Alex Abbas for his advice regarding analysis of microarray data, and Laura Sanders for assistance with figures.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
All authors are current or former employees of Genentech, Inc. No currently marketed Genentech products are mentioned in this manuscript but it is possible the research could contribute to future commercial products or impact other drug development processes.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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{at}gene.com Back

2 Abbreviations used in this paper: IEC, intestinal epithelial cell; IBD, inflammatory bowel disease; KLH, keyhole limpet hemocyanin; LCM, laser capture microscopy; siRNA, small interfering RNA. Back

Received for publication August 2, 2007. Accepted for publication January 9, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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