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The Journal of Immunology, 2007, 179: 7012-7020.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Resistin-Like Molecule β (RELMβ/FIZZ2) Is Highly Expressed in the Ileum of SAMP1/YitFc Mice and Is Associated with Initiation of Ileitis1

Sean L. Barnes2,*, Alda Vidrich2,*, Mei-Lun Wang{dagger}, Gary D. Wu{dagger}, Fabio Cominelli*, Jesus Rivera-Nieves*, Giorgos Bamias* and Steven M. Cohn3,*

* Digestive Health Center of Excellence, University of Virginia, Charlottesville, VA 22908; and {dagger} Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
SAMP1/Fc mice develop spontaneous ileitis that shares many features with human Crohn’s disease. One of the earliest features of ileitis in SAMP1/Fc mice is an increase in the number of ileal goblet and intermediate cells. Resistin-like molecule β (RELMβ) is a goblet cell-specific, cysteine-rich peptide previously shown to function as part of the innate immune response. In this study, we examined the role of expression of RELMβ in the initiation of ileal inflammation in SAMP1/Fc mice. RELMβ was highly induced in the ilea of SAMP1/Fc mice beginning at age 5 wk, coincident with the histological appearance of inflammation. RELMβ was found in ileal goblet cells and some intermediate and Paneth cells. Surprisingly, RELMβ mRNA levels were significantly increased in the ilea of 80% of germ-free SAMP1/Fc mice examined compared with specific pathogen-free AKR control mice of similar age. Ileitis was observed in germfree SAMP1/Fc mice, although it was attenuated relative to specific pathogen-free SAMP1/Fc mice. These data suggest that neither the early induction of RELMβ expression nor ileal inflammation requires the presence of viable intestinal flora. Neither was the induction of RELMβ dependent on the major Th1 or Th2 cytokines. However, RELMβ stimulated naive bone marrow-derived macrophages to secrete significant amounts of TNF-{alpha}, IL-6, and RANTES. Our data suggest that RELMβ is involved in the initiation of ileitis in SAMP1/Fc mice and may act through the induction of proinflammatory cytokines from resident immune cells within the mucosa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The SAMP1/Yit mouse is a recombinant-inbred line that spontaneously develops ileitis. Matsumoto et al. (1) originally derived this mouse strain from AKR mice. The SAMP1/YitFc (SAMP1/Fc) substrain originated from SAMP1/Yit mice following 20 generations of brother-sister mating in our colony at the University of Virginia (2). This substrain is distinguished from the parental SAMP1/Yit by the emergence of perianal fistulizing disease, a pathological feature that has not been reported in the original SAMP1/Yit colony. SAMP1/Yit and SAMP1/Fc mice develop a spontaneous ileitis that is similar in many features to human Crohn’s disease. Development of ileitis in these mice is accelerated by the presence of luminal bacteria and is characterized by discontinuous segmental inflammation involving the ileum while sparing the proximal small intestine and colon (1, 2). Histopathologic features of ileitis in this model include transmural inflammation, crypt abscesses, and epithelial changes including loss of villi, crypt elongation, and crypt branching. The spontaneous onset of inflammation is nearly 100% by 10 wk of age.

The intestinal epithelium is the major interface between the luminal environment and the host, acting as a barrier to regulate the uptake and presentation of luminal Ags and bacteria to the mucosal immune system. The intestinal epithelium is a vital component of the innate immune response as well, and plays an active role in host defense against luminal bacteria and other pathogens. For example, Paneth cells and goblet cells elaborate a variety of antimicrobial peptides and other mediators in the normal intestine. Paneth cells seem to have direct antimicrobial activity through the secretion of peptides such as defensins/cryptdins as well as enzymes such as lysozyme and phospholipase A2 that can dissolve bacterial cell walls and other components (3, 4, 5). In contrast, mucin and intestinal trefoil factor 3 (Tff3),4 products of mature goblet cells, prevent interactions of the epithelium with luminal bacteria, and facilitate epithelial restitution, ensuring that the barrier function of the intestinal epithelium is maintained (6, 7, 8). We have recently reported an expansion of the goblet, intermediate, and Paneth cell populations in the ilea of SAMP1/Fc mice. Increased numbers of these cells within elongated crypts are seen as early as 4 wk of age, a time when there is no histological evidence of ileitis. These cell populations further expand with increasing age and degree of inflammation (9).

Resistin-like molecule β (RELMβ), also known as mouse found in inflammatory zone 2 (mFIZZ2), is a cysteine-rich secreted protein that under normal noninflamed conditions is highly expressed in the murine colon but is only minimally expressed in the small intestine (10, 11). Undifferentiated crypt epithelial cells and goblet cells in the colon express RELMβ mRNA, while the protein is found in goblet cell mucin granules and is also detected at high levels in both mouse and human stool (11, 12). Expression of the related RELM{alpha} gene is enhanced in OVA-induced pulmonary inflammation in BALB/c mice (10), a finding that first suggested an association between the expression of RELM proteins and the inflammatory response. Although the specific functional role of RELMβ has not been established, its up-regulation in the colon by conventional colonic flora together with the finding that it is induced by intestinal nematode infection suggest that RELMβ also may function as an immune effector molecule (12, 13). Strong support for this hypothesis comes from the recent demonstration that RELMβ–/– mice do not develop the classic fulminant acute colitis associated with dextran sulfate sodium (DSS) treatment (14, 15). Rather, the absence of colonic RELMβ expression in the RELMβ–/– mice affords protection from DSS-induced acute inflammation. It is currently not known whether this goblet cell-specific protein participates in regulating the onset of chronic intestinal inflammation such as is seen in inflammatory bowel disease (IBD). Therefore, in this study, we examined whether RELMβ is expressed in the ilea of SAMP1/Fc mice, determined its relationship to the onset of spontaneously occurring chronic ileitis seen in these mice, and examined whether RELMβ can modulate proinflammatory immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and treatments

The SAMP1/YitFc mice (SAMP1/Fc) mice are a substrain derived from SAMP1/Yit mice provided by S. Matsumoto of the Yakult Central Institute for Microbiological Research (Tokyo, Japan). C57BL/6J and AKR mice, used as controls, were obtained from The Jackson Laboratory. TNF{Delta}ARE/wt mice were reared under specific pathogen-free (SPF) conditions as previously described (16), backcrossed to C57BL/6J for 20 generations, and a colony of these mice is maintained at the University of Virginia. All mice were housed in autoclaved microisolator cages maintained under positive pressure HEPA-filtered air in a SPF facility at the University of Virginia. Germfree (GF) SAMP1/Fc mice were derived from SAMP1/Fc breeding stock (obtained from the University of Virginia colony) at Taconic laboratories and were maintained at Taconic Farms. Germfree, or axenic, animals were maintained in flexible-film or semirigid isolators. GF mice were tested using the International Health Monitoring System and were free of all aerobic and anaerobic organisms. A complete list of agents that were tested for in GF SAMP1/Fc mice can be obtained from the Taconic website (www.taconic.com/healthr/healthstandards.htm). Control mice were maintained on a strict 12-h light/dark cycle, received autoclaved tap water supplied in individual sipper bottles, and were fed autoclaved standard laboratory chow ad libitum.

Some mice were treated for 3 wk with an antibiotic mixture (30 ml/two mice/cage) that substituted for their drinking water formulated to contain ciprofloxacin, metronidazole, vancomycin, and imipenem (Island Pharmacy Services), each at 0.15 mg/ml in distilled water containing sucrose (28.3% w/v). Sucrose was included to render the antibiotic solution palatable. Control mice received distilled water containing only sucrose (28.3% w/v; Island Pharmacy Services). Antibiotic and vehicle solutions were changed daily and consumption measured. Animal bedding and cages were changed daily to prevent reinfection of animals. There were no differences in liquid consumption between mice given antibiotic mixture and those receiving the sucrose solution. Mice were 3 wk of age at the beginning of antibiotic treatment. There was no significant difference between weights of mice in the antibiotic-treated and vehicle control groups. At 6 wk of age, mice were euthanized, the whole intestinal tract was quickly removed, divided into intestinal segments, and each segment was snap-frozen and stored in liquid nitrogen until RNA isolation and PCR analysis. Other groups of 3- to 4-wk-old SAMP1/Fc mice were administered either 0.5 mg of anti-IFN-{gamma} mAb (clone XMG1.2; DNAX Laboratories) by tail vein injection weekly for 5 wk, 1.0 mg of anti-mouse IL-4 mAb by i.p. injection every 5 days for a total of 10 injections, or 1.0 mg of anti-mouse chimeric IL-4R-M1 Ab, previously shown to effectively block IL-4R-dependent responses (provided by Amgen) (17, 18), twice a week for 3 wk. All mice used in these studies were cared for in accordance with protocols approved by the University of Virginia Institutional Animal Care and Use Committee, using Association for Assessment of Laboratory Animal Care guidelines.

Histology and immunohistochemistry methods

Tissue from the indicated regions of the small intestine was rapidly dissected from mice, fixed in either Bouin’s fixative or neutral-buffered formalin, and embedded in paraffin as previously described (9). For analysis of inflammation, the distal 15 cm of small intestine was fixed in Bouin’s fixative. Deparaffinized 3-µm sections were stained with H&E, and graded for the severity and extent of villus distortion, acute inflammation (granulocyte infiltration), and chronic inflammation (mononuclear cell infiltration). For each parameter, tissue samples were scored from 0 (normal) to 3 (severe). This severity score was then multiplied by a factor representing the extent of inflammation, which ranged from 0.5 (single focus of inflammation) to 4 (75% involvement of the mucosal surface area). A total inflammatory index was obtained by adding the individual scores for each category. Periodic acid-Schiff (PAS) Alcian-blue staining identified goblet cells, whereas Paneth and intermediate cells, identified by their characteristic eosinophilic granules, were enumerated in neutral-buffered formalin-fixed tissue sections. For immunohistochemistry, deparaffinized tissue sections were incubated with affinity purified murine RELMβ polyclonal Ab as described by Wu and colleagues (12).

Quantification of mRNA levels

Levels of mRNA for RELMβ in mouse intestinal segments (proximal jejunum, ileum, and colon) were quantified using real-time PCR analysis. Total RNA was isolated from each sample using the RNeasy kit from Qiagen according to the manufacturer’s directions. cDNA was prepared by reverse transcription of the isolated total RNA and mRNA levels were quantified by real-time PCR using an ABI PRISM SDS7000 sequence detection system (Applied Biosystems). For reverse transcription, random hexamers (1 µg) and either 10 or 50 ng of total RNA were used in a final reaction volume of 20 µl containing 200 U of Superscript (Invitrogen Life Technologies). Primers for PCR were designed using the Primer Express software (Applied Biosystems) and the sequence data found in GenBank. Primers for measuring levels of RELMβ mRNA were: RELMβ forward primer, 5'-GGCTGTGGATCGTGGGATAT-3' and reverse primer, 5'-GAGGCCCAGTCCATGACTGA-3'. The PCR was performed in triplicate using 10% of the volume of the first strand synthesis in a total volume of 25 µl that included ~8 µl of SYBR Green core reagents (Applied Biosystems) and 250 nM final concentration of primers. Primer concentrations were previously optimized as suggested in User Bulletin no. 2 (Applied Biosystems). The amplification of cDNA was performed for 40 cycles by a preset cycling program that included the generation of a melting curve. Thermocycling conditions were as follows: 1) 50°C for 2 min (activation of AmpErase UNG); 2) 95°C for 10 min (activation of AmpliTaq Gold enzyme); 3) 95°C for 15 s (denaturation) and 60°C for 1 min (anneal/extend) for 40 cycles. PCR controls included "no RT" and "no template" reactions. The {Delta}CT (cycle threshold) method was used to quantitate relative mRNA levels as described in User Bulletin no. 2 (Applied Biosystems) using18s RNA as the reference and internal standard. TaqMan primer-probe set for 18s RNA with the VIC/TAMRA detection system was used to simultaneously measure 18s RNA in replicate samples. IL-4 and IL-13 mRNA levels were quantified using Applied Biosystem’s Celera Assay-on-Demand kit according to the manufacturer’s directions.

Determination of cytokine levels

Bone marrow (BM) cells extracted from the femurs and tibias of 10- to 11-wk-old male AKR and SAMP1/Fc mice were cultured for 5 days in DMEM supplemented with heat-inactivated FBS (FBS–10%) (Mediatech), and L929 cell-conditioned medium (20%). On day 6, cells were transferred to 24-well plates at a concentration of 5 x 105 cells/well. Cells were allowed to attach overnight and on the following day were treated for 48 h with rRELMβ (PeproTech) or LPS (Sigma-Aldrich) diluted in DMEM supplemented with only 10% FBS. The amounts of LPS used were based on the endotoxin level (<80 pg/µg) of the lot of RELMβ that was used, as reported by the manufacturer. Before treating some cells, RELMβ and LPS were incubated for 30 min at 37°C with polymyxin B (Sigma-Aldrich), at a final concentration of 50 µg/ml. Cytokines secreted in the medium were quantified by the Beadlyte multicytokine detection kit (Upstate) and the Luminex suspension array detection system.

Statistical methods

Data were analyzed by pairwise t tests using the pooled estimate of variance and Bonferroni’s correction of the p values for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RELMβ is increased in the ileum of SAMP1/Fc mice with inflammation

RELMβ mRNA levels were measured in the ileum of SAMP1/Fc and control mice at a time point before the onset of inflammation and at a time point when ileal inflammation is fully established in the SAMP1/Fc mice. At 4 wk of age, when no ileal inflammation is histologically evident in SAMP1/Fc mice, very low but detectable levels of RELMβ mRNA were present in the ileum of all three mouse strains. By 10 wk of age, a time when marked inflammation is typically evident, RELMβ mRNA level in the SAMP1/Fc ileum was increased ~3 orders of magnitude over the level seen at 4 wk (Fig. 1). By contrast, ileal RELMβ expression in AKR and C57BL/6J (B6) mice, which do not develop spontaneous inflammation, showed only a small increase between ages 4 and 10 wk. In AKR mice, this increase was statistically significant, although the magnitude of this response was much smaller than that observed in SAMP1/Fc ileum and did not correlate with ileal RELMβ protein expression. At age 10 wk, levels of RELMβ mRNA in the ilea of SAMP1/Fc were increased ~140- and ~3,000-fold compared with levels observed in the ilea of age-matched AKR and B6, respectively.


Figure 1
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  		FIGURE 1. RELMβ is highly induced in the ilea of SAMP1/Fc mice. Expression of RELMβ mRNA was determined in the ilea of SAMP1/Fc mice and control mouse strains (AKR, C57BL/6J (B6)) by quantitative real-time PCR analysis. Levels of RELMβ mRNA were determined at an age before the onset of inflammation in the SAMP1/Fc mouse (4 wk) or at an early time point after inflammation was established (10 wk). Data are expressed as the mean ± SD RELMβ mRNA levels are expressed relative to 18s rRNA (n = 4/age group).

 
Regional expression of RELMβ

RELMβ mRNA levels were measured in the proximal jejunum, ileum, and colon of SAMP1/Fc mice and in the same intestinal segments of AKR and B6 control mice, at 10 wk of age. RELMβ was not equally expressed in all regions of the intestine. RELMβ mRNA levels were low to barely detectable in the proximal jejunum for all mouse strains examined. RELMβ mRNA in SAMP1/Fc ileum was found to be at least 2 orders of magnitude greater than that in either AKR or B6 ileum and ~130-fold higher than the level reached in the SAMP1/Fc proximal jejunum (Fig. 2). RELMβ mRNA levels in the ileum of SAMP1/Fc mice approached those seen in the colon of noninflamed AKR and B6 controls. Consistent with previous reports, the highest level of intestinal RELMβ expression was in the colon of the three mouse strains examined. Nevertheless, colonic RELMβ mRNA levels of SAMP1/Fc mice were still 11- and 24-fold greater than those found in either AKR or B6 mice, respectively (Fig. 2).


Figure 2
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  		FIGURE 2. Regional expression of RELMβ in the SAMP1/Fc mouse intestinal tract. RELMβ mRNA levels were determined by quantitative real-time PCR in the jejunum, ileum, and colon of 10-wk-old SAMP1/Fc mice, AKR and B6. Data are expressed as the mean ± SD RELMβ mRNA levels are expressed relative to 18s rRNA (n = 4/age group).

 
Cell-specific expression of RELMβ

SAMP1/Fc mice develop segmental ileitis by age 10 wk with nearly 100% penetrance (1, 2). Segmental ileitis in these mice is characterized by a mixed inflammatory infiltrate and histopathological evidence of epithelial injury, including villus shortening, cryptitis, and formation of crypt abscesses (2, 19). Dramatic increases in the number of goblet cells, intermediate cells, and Paneth cells within elongated crypts were seen in the ilea of SAMP1/Fc mice as previously reported (9). Cells with Paneth cell-type granules were found distributed throughout the crypt up to the crypt-villus junction and occasionally on the lower portion of the villus epithelium. Many of these granule-containing crypt epithelial cells were positive for staining with PAS-Alcian Blue, suggesting that these cells also express mucin, a finding consistent with an intermediate cell phenotype (9). In an initial recent study, we reported that immunoreactive RELMβ was detectable in ileal goblet cells of 40-wk-old SAMP1/Fc mice with longstanding ileitis (14, 15). We now have extended this observation by examining the cellular distribution of RELMβ in the ileum of younger SAMP1/Fc mice during the initial stages of ileitis in this mouse strain. By age 10 wk, immunoreactive RELMβ was prominently expressed in most of the goblet cells, as well as in some intermediate and Paneth cells located at the base of the ileal crypt epithelium in SAMP1/Fc mice (Fig. 3, B and C). By contrast, immunoreactive RELMβ was not detected in any cell type in the ileum of 10-wk-old AKR controls (Fig. 3A) and was only occasionally observed in isolated crypt base epithelial cells at older ages in these control mice (data not shown).


Figure 3
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  		FIGURE 3. Cellular localization of RELMβ in the ileum of SAMP1/Fc and AKR mice. Sections of ileum from 10-wk-old mice were analyzed by immunohistochemistry with an Ab to mouse RELMβ (B and C). No RELMβ staining is evident in the ileum of AKR controls (A). In the SAMP1/Fc ileum, prominent immunoreactive staining is noted in most goblet cells in areas of ileal inflammation (open arrows, B). C, A higher magnification of the crypt base region bounded by the rectangle in B. Note the presence of immunoreactive RELMβ in granule-containing cells at the crypt base, which are likely Paneth or intermediate cells (solid arrows, B and C). Some cells that appear undifferentiated also stain in mid-crypt. mRNA levels for goblet cell-specific genes, Muc2 and Tff3, were determined by quantitative real-time PCR in SAMP1/Fc and in AKR ileum and are expressed relative to18S rRNA (D and E).

 
Because we observed a greater than normal number of goblet and intermediate cells in the ilea of 10-wk-old SAMP1/Fc mice, the increase in RELMβ mRNA levels could simply be reflective of this increase in cell number or due to a nonspecific global up-regulation of goblet/intermediate cell-specific genes. Therefore, we examined the expression of two other goblet cell-specific genes, Muc2 and Tff3, and found no significant difference in their expression in the ilea of SAMP1/Fc mice between 4 wk (before any histologic evidence of ileitis) and 10 wk of age (established ileitis) (Fig. 3, D and E). Additionally, there was no significant difference in either Muc2 or Tff3 expression between SAMP1/Fc mice and controls. Therefore, the specific induction of RELMβ is not merely a consequence of the dramatic expansion of the goblet and intermediate cell lineages seen in the inflamed ilea of SAMP1/Fc mice.

Initial induction of RELMβ expression in the ileum of SAMP1/Fc mice is coincident with the appearance of ileal inflammation

To determine the temporal relationship of RELMβ expression to the onset of inflammation, we determined both the level of RELMβ mRNA and inflammatory index of the ilea of SAMP1/Fc mice and AKR controls at various ages, starting at age 3 wk. Expression of RELMβ mRNA was dramatically induced beginning at 5 wk of age (Fig. 4A). Histologic evidence of ileal inflammation was first observed in SAMP1/Fc mice between ages 5 and 6 wk, coincident with the rapid rise in RELMβ mRNA levels (compare Fig. 4). The high level of ileal RELMβ mRNA was sustained from age 5 to 10 wk in SAMP1/Fc mice. However, the inflammatory index of SAMP1/Fc ilea, which is a composite measure that includes both acute and chronic inflammation, peaked at 8 wk and remained high through 10 wk of age. By contrast, the induction of RELMβ mRNA levels seen in the ilea of AKR control mice, between age 5 to 6 wk, was about ~100-fold less than that observed in SAMP1/Fc ilea and was not accompanied by a significant concomitant increase in the ileal inflammatory score.


Figure 4
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  		FIGURE 4. Expression of RELMβ is induced in the ileum of SAMP1/Fc mice, at or before the onset of inflammation. RELMβ mRNA levels were determined by quantitative real-time PCR in the ileum of SAMP1/Fc mice at the indicated ages (n = 4–6/age group) (A). Inflammatory scores were determined histologically in ileal sections of SAMP1/Fc mice at the same ages, as previously described (2 ) (n = 4–13 mice/age group) (B). Data are expressed as the mean ± SD RELMβ mRNA level are expressed relative to 18s rRNA.

 
Viable luminal flora is not required for the induction of ileal RELMβ expression in SAMP1/Fc mice

Prior studies have demonstrated that RELMβ expression in the colon is regulated by bacterial colonization (12). We therefore examined whether the presence of luminal flora was required for the induction of RELMβ expression in the ilea of SAMP1/Fc mice. Starting at 3 wk of age, well before any clinical signs of ileal inflammation, SAMP mice were treated for 3 wk with an antibiotic mixture demonstrated to effectively reduce to undetectable levels the component of the intestinal bacterial content that can be assessed by culture of fecal samples (20). The effect of this antibiotic treatment on RELMβ expression was then examined in intestinal tissue harvested from the treated and control 6-wk-old SAMP1/Fc mice. Oral administration of the antibiotic mixture did not prevent the induction of high levels of RELMβ expression that is seen at age 6 wk and this expression was not different from untreated SAMP1/Fc control mice (Fig. 5A). Additionally, antibiotic treatment did not prevent the appearance of the early manifestations of inflammation that are evident in these mice at age 6 wk (total inflammatory score; treated, 5.75 ± 4.18 vs control, 6.17 ± 2.48).


Figure 5
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  		FIGURE 5. Effect of luminal flora on induction of ileal RELMβ mRNA expression in SAMP1/Fc and control mice. Ileal RELM-β mRNA expression in 6-wk-old SPF SAMP1/Fc mice following 3 wk of oral antibiotic treatment consisting of a mixture of ciprofloxacin, metronidazole, vancomycin, and imipenem as given in Materials and Methods (A). Expression of RELMβ mRNA was determined by quantitative real-time PCR in ileal total RNA samples. RELMβ mRNA levels are expressed relative to 18s rRNA (n = 5–8 mice/group). Data are expressed as the mean ± SD. Expression of RELMβ mRNA was determined at the ages indicated by quantitative real-time PCR in total ileal RNA from GF SAMP1/Fc mice, SAMP1/Fc mice raised in SPF housing, and AKR controls raised in SPF housing (B). RELMβ mRNA levels are expressed relative to 18s rRNA (n = 5–6 mice/age group). Data for SPF SAMP1/Fc mice (•) and AKR mice ({square}) are expressed as the mean ± SD. {circ}, RELMβ mRNA levels for each of 12 GF SAMP1/Fc mice analyzed.

 
Much of the luminal bacterial flora is not detected by standard microbiological techniques. Because the antibiotic treatment protocol reduces, but does not eliminate, the intestinal flora of treated mice, we also measured RELMβ expression in the intestines of SAMP1/Fc mice raised under GF conditions. Among 10- to 40-wk-old GF SAMP1/Fc mice, RELMβ mRNA levels were still significantly higher in the ileum of 80% of mice examined relative to the SPF AKR controls (Fig. 5B) and were modestly decreased compared with the levels observed in the ileum of SPF SAMP1/Fc counterparts. An additional and unexpected finding was the presence of significant ileal inflammation in these GF SAMP1/Fc mice, although the inflammation was attenuated relative to SPF SAMP1/Fc mice (6.5 ± 0.7 vs 15.2 ± 1.3, p < 0.0001). Additionally, RELMβ mRNA levels in the ileum of SPF SAMP1/Fc mice continued to increase during the chronic phase of inflammation (after age 10 wk), and such an increase was not seen in age-matched AKR controls (Fig. 5B). Finally, similar to the expression of RELMβ in the colon of SPF SAMP1/Fc mice (see Fig. 2), RELMβ mRNA levels in the colon of GF SAMP1/Fc mice were also higher than those found in the colon of adult AKR mice (data not shown).

The initial induction of ileal RELMβ expression is not dependent on the expression of Th1 cytokines

Elevations in expression of Th1 cytokines, IFN-{gamma} and TNF-{alpha}, have been previously observed in SAMP1/Fc mice as early as 4 wk of age as compared with control mice (2, 21). In view of our previous findings, we examined whether the Th1 cytokines, IFN-{gamma} and TNF-{alpha}, could regulate the induction of RELMβ in the ileum (Fig. 6). To determine whether IFN-{gamma} regulates expression of RELMβ, SAMP1/Fc mice were treated for 5 wk, beginning at 3 wk of age, with monoclonal neutralizing anti-IFN-{gamma} Ab. A small but significant decrease in inflammatory scores was achieved by this treatment but the ileal expression of RELMβ was not decreased (Fig. 6A). To determine whether TNF-{alpha} might drive expression of RELMβ, we used mice genetically engineered to overexpress TNF-{alpha} due to the deletion of the TNF AU-rich elements (ARE) (16). Mice carrying the TNF {Delta}ARE mutations develop a Crohn’s-like ileitis driven by the constitutive overproduction of TNF-{alpha} due to deletion of the ARE. When we compared RELMβ expression of TNF{Delta}ARE/wt with that of wild-type littermates, the TNF{Delta}ARE/wt mice did not have increased levels of ileal RELMβ mRNA at either 4 or 10 wk of age (Fig. 6B) indicating that elevated TNF-{alpha} levels do not drive RELMβ expression in this model of intestinal inflammation.


Figure 6
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  		FIGURE 6. Relationship of Th1 cytokines to RELMβ expression in SAMP1/Fc mice. RELMβ mRNA levels and inflammatory scores were determined as previously described in Fig. 4. Levels of mRNA are expressed relative to 18s rRNA. SAMP1/Fc mice were treated with anti-IFN-{gamma} mAb or an isotype control (A). Data are expressed as the mean ± SD (n = 6–8 mice/group). RELMβ mRNA levels and inflammatory scores were determined in TNFwt/wt and TNF{Delta}ARE/wt mice (B). RELMβ mRNA levels are normalized to the 10-wk wt/wt value. Data are expressed as the mean ± SD (n = 4 mice/group).

 
The induction of ileal RELMβ expression is not dependent on Th2 cytokines

Previous studies have implicated Th2 cytokines in the regulation of colonic RELMβ expression (12). Therefore, the time courses of expression of two Th2 cytokines were examined in the ilea of SAMP1/Fc mice and their age-matched controls. A small increase (3.9-fold higher than that seen in AKR controls) in IL-4 mRNA levels was observed at 4 wk of age in SAMP1/Fc mice, just before the observed induction of RELMβ (Fig. 7A). To examine the possibility that this early increase in ileal IL-4 might drive the induction of RELMβ, we blocked the activity of this cytokine by treating SAMP1/Fc mice with a neutralizing mAb to mouse IL-4. RELMβ mRNA levels were not significantly altered as a consequence of treatment with anti-IL-4 mAb (Fig. 7B) nor was there a significant change in ileal inflammation (data not given). In contrast to IL-4, the highest expression of IL-13 was attained by age 16 wk, at which time the levels of mRNA were 111-fold higher than the levels seen in AKR controls. Nevertheless, to eliminate the possibility that even low levels of IL-13 might induce RELMβ expression in the ileum of SAMP1/Fc mice, we blocked IL-13 and IL-4 activity via blocking IL-4R{alpha} signaling by treating mice with a chimeric rat Ab to the {alpha} subunit of the IL-4R (IL-4R{alpha}). IL-13 activity depends on the formation of a high-affinity receptor-signaling complex between IL-13R{alpha}1 and IL-4R{alpha}, which also binds IL-4 (22). Anti-IL-4R{alpha} treatment did not abrogate the induction of RELMβ mRNA that occurs by age 5 wk. However, a small reduction was seen in the ultimate levels of RELMβ mRNA attained in the ilea of anti-IL-4R{alpha}-treated SAMP1/Fc mice compared with those of untreated control SAMP1/Fc mice (Fig. 7B).


Figure 7
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  		FIGURE 7. Relationship of Th2 cytokines to RELMβ expression in SAMP1/Fc mice. Levels of mRNA for IL-4 and IL-13 were determined at the ages indicated by quantitative real-time PCR of RNA isolated from ilea of SPF SAMP1/Fc mice and AKR controls (A). Levels of mRNA are expressed relative to 18s rRNA. Data are expressed as the mean ± SEM (n = 4–6 mice/age group). SAMP1/Fc mice were treated with anti-IL-4 mAb (IL-4 neutralization), isotype control, or anti-IL-4R (IL-4/IL-13 blockade) and results of these treatments are compared with pretreatment control mice at ages 3–4 wk (B). RELMβ mRNA levels were determined as given in Fig. 4. Data are expressed as the mean ± SD (n = 6–8 mice/group).

 
RELMβ stimulates macrophages to produce proinflammatory cytokines

Because the appearance of ileitis in SAMP1/Fc mice is coincident with the induction of RELMβ at 5–6 wk of age, we examined whether RELMβ can stimulate cells of the adaptive immune system to produce cytokines that may initiate this inflammation. Cultured naive BM-derived macrophages from AKR mice secreted substantial levels of TNF-{alpha} and IL-6 as well as the chemokine, RANTES, in response to the addition of rRELMβ to the culture medium (Fig. 8A). Secretion of TNF-{alpha}, IL-6, and RANTES by AKR BM macrophages showed a dose-dependent response to concentrations of RELMβ from 0 to 10 µg/ml (Fig. 8B). The levels of TNF-{alpha} attained in the culture medium of AKR and SAMP1/Fc macrophages in response to treatment with RELMβ were not significantly different from each other suggesting that the inherent sensitivity of BM-derived macrophages to RELMβ between AKR and SAMP1/Fc mice is not different (Fig. 8C).


Figure 8
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  		FIGURE 8. Effect of rRELMβ on BM-derived macrophages. BM-derived macrophages from 10- to 11-wk-old AKR mice were treated with rRELMβ (2 µg/ml final concentration), pretreated with polymyxin B (PXB), for 48 h and the levels of TNF-{alpha}, IL-6, and RANTES secreted into the medium measured as given in Materials and Methods (A). Control cells were either untreated, treated with LPS or treated with LPS preincubated for 30 min with PXB (50 µg/ml). PXB treatment suppressed LPS-induced cytokine production by macrophages to levels that were the same as that of untreated control cells. Data are expressed as the mean ± SE for triplicate samples per treatment group. Dose response of macrophages to rRELMβ (final concentrations ranging from 0 to 10 µg/ml) previously incubated with PXB as given above isolated from 10- to 11-wk-old AKR mice (B). Triplicate samples were analyzed for each treatment group, and data are expressed as the mean ± SEM. Levels of TNF-{alpha} secreted by macrophages isolated from 10- to 11-wk-old AKR and SAMP1/Fc mice following incubation with 10 µg/ml RELMβ (PXB treated) (C). Data are expressed as the mean ± SD for triplicate samples per treatment group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that the early and rapid induction of ileal RELMβ expression is associated with the onset and development of inflammation in SAMP1/Fc mice, a model of spontaneous chronic ileitis. Within the small intestine, the induction of RELMβ mRNA and increased protein expression occurred only in the ileum, the predominant site of inflammation in SAMP1/Fc mice. Ileal RELMβ mRNA levels increased ~2,000-fold between 4 and 6 wk of age, coincident with the earliest histologic evidence of ileal inflammation in SAMP1/Fc mice, suggesting a linkage between induction of RELMβ and the initial events in the pathogenesis of inflammation in this model of spontaneous chronic ileitis. Our demonstration that RELMβ induction is an early manifestation of inflammation and is not dependent on luminal flora or Th1 and Th2 cytokines raises the possibility that induction and expression of RELMβ may promote inflammation and/or mucosal injury under appropriate pathological circumstances. This hypothesis is further supported by recent studies that show that RELMβ can also modulate inflammation in DSS-induced colitis, a model of acute inflammation (14, 15).

One of the earliest histopathologic features associated with the onset of ileitis in SAMP1/Fc mice is an expansion of the goblet, intermediate, and Paneth cell lineages in the inflamed intestine of these mice (9). RELMβ protein was observed in goblet cells as well as in some granule-containing cells residing at the crypt base in the inflamed ilea of SAMP1/Fc mice. Many of these cells located at the crypt base also contained mucins, suggesting that they might be intermediate cells that display features of both goblet and immature Paneth cells. This restricted cellular distribution of RELMβ is in agreement with earlier reports (10, 11). Despite the expansion of these goblet cells and the high level of cellular RELMβ expression observed in these cells, other goblet cell-specific genes (i.e., Tff3 and Muc2) were not up-regulated. This finding indicates that these hyperplastic goblet cells have a unique phenotype with respect to the spectrum of peptides they produce and raises the possibility that they may be functionally different from goblet cells of normal noninflamed ileum. The rapid induction of ileal goblet cell RELMβ expression that we observe in this study and its temporal association with the onset of ileitis in SAMP1/Fc mice further point to goblet cell products, including RELMβ, as possible key mediators in the pathogenesis of Crohn’s ileitis. These observations are consistent with the emerging concept that goblet cells have an active role in innate immunity (3, 23, 24, 25).

The dramatic induction of RELMβ that we observed in the ileum of SAMP1/Fc mice, between ages 4 and 6 wk, was not driven by the Th1 and Th2 cytokines thought to be involved in the pathogenesis of ileitis in this mouse model. Levels of RELMβ mRNA in SAMP1/Fc ilea were not significantly reduced by treatment with anti-IFN-{gamma} Ab nor were levels of RELMβ increased in the ileum of TNF{Delta}ARE/wt mice, a murine model in which excessive TNF production drives the onset of ileitis. In a previous study, RELMβ expression in both the colon and the small intestine, in response to nematode infection, was thought to be regulated by Th2 cytokines (13). However, in this study, we find that Th2 cytokines do not regulate the initial induction of RELMβ in SAMP1/Fc ileum. Blockade of IL-4 and IL-13 type II receptor complex signaling did not abolish the induction of ileal RELMβ in SAMP1/Fc mice. The small reduction in the levels of RELMβ expression in the ilea of SAMP1/Fc mice treated with the anti-IL-4R{alpha} Ab is consistent with the prior demonstration that Th2 cytokines can regulate the levels of RELMβ expression as seen in helminth infections of the intestine (13). The establishment of chronic ileitis and persistent high expression of RELMβ did coincide with the significant increase in IL-13 mRNA expression that we observed in the ilea of SAMP1/Fc mice during chronic inflammation that occurs after 8–10 wk of age. Thus, while the type II receptor signaling complex, that is formed in response to both IL-13R and IL-4R binding, is not involved in the initial induction of RELMβ in SAMP1/Fc ilea, it remains possible that IL-13 modulates the ultimate steady-state levels of RELMβ expression observed when chronic inflammation predominates.

The observed presence of ileal inflammation in GF SAMP1/Fc mice stands in contrast to many other animal models of IBD, which require the presence of enteric flora for the development of inflammation (26). The current observations that SAMP1/Fc mice, derived and maintained in a GF environment, show significant induction of ileal RELMβ compared with SPF AKR mice and spontaneously develop moderate ileal inflammation suggest that bacterial colonization is not solely responsible for either the induction of expression of RELMβ or the initiation of ileitis in these mice. Our latter observation is consistent with a recent study showing that while commensal bacteria are not necessary for the development of ileitis in SAMP1/Fc mice, the presence of bacteria results in a more severe intestinal inflammation (27). Similarly, while induction of RELMβ expression is independent of viable gut flora, it is likely that bacteria may have some effect on regulating the levels of RELMβ chronically present in the ileum of SAMP1/Fc mice given our observations that RELMβ expression in the ileum of GF SAMP1/Fc mice was modestly reduced from that seen in the SPF SAMP1/Fc. Nevertheless, RELMβ levels in the ilea of GF SAMP1/Fc mice were still elevated relative to those of SPF AKR controls.

Interestingly, elevated levels of RELMβ mRNA were also found in the colon of both GF and SPF SAMP1/Fc mice, although these animals do not develop colitis. These data suggest that the regulation of RELMβ, in SAMP1/Fc mice, is different from that reported in other mouse strains (12). One possibility is that other antigenic factors contribute to inflammation and to RELMβ expression in SAMP1/Fc mice such as luminal Ags previously implicated in the pathogenesis of Crohn’s disease (28, 29, 30, 31). Alternatively, the increase in RELMβ expression may, in part, be a genetically determined trait in SAMP1/Fc mice such that either the baseline level of expression is higher or there is over-expression upon Ag stimulation. These possibilities are suggested by two observations: 1) the colonic expression of RELMβ in SAMP1/Fc mice is higher than that seen in control mice in the absence of any colonic inflammation; and, 2) the colon of GF SAMP1/Fc mice also expressed significant levels of RELMβ mRNA.

Although the precise function of RELMβ is unknown, our findings along with a number of recent studies suggest that RELM proteins, including RELMβ, play a role in regulating inflammatory responses in the intestine and other mucosa. For example, Holcomb et al. (10) observed enhanced expression of RELM{alpha} (mFIZZ1) in a mouse model of allergic pulmonary inflammation. This group suggested that enhanced RELM{alpha} expression mediates the interactions between bronchial tissue, inflammatory cells, and the visceral sensory organs in allergic inflammation. More recent studies demonstrate that both RELM{alpha} and RELMβ are early induced gene products in lungs of Ag-challenged mice (32). Increased RELMβ expression is also observed in the small intestinal mucosa of the CFTR-null mouse, a model of cystic fibrosis (33). The CFTR-null mouse displays a severe intestinal phenotype that includes mucosal inflammation and up-regulation of components of the innate immune system. In this study, RELMβ shows the highest induction of all the genes from the small intestine represented in the microarray analysis, suggesting a role for this protein/gene in inflammation. Expression of RELMβ is highly induced in both the colon and the small intestine in a mouse model of nematode infection and enhanced clearance of worms associated with the induction of a Th2-mediated inflammatory response (13).

The early induction of RELMβ expression relative to the appearance of inflammation, as well as the independence of ileal RELMβ induction from key Th1 and Th2 cytokines and commensal bacterial colonization, suggests that RELMβ may be an important mediator involved in the initiation of ileitis in the SAMP1/Fc mouse model of IBD. Very recent studies support a role for RELMβ as a proinflammatory molecule in the intestine. Mice with a RELMβ gene disruption are relatively resistant to both the clinical and histological manifestations of acute DSS-induced colitis (14, 15). These data raise the question of how luminally secreted RELMβ might gain access to cells of the mucosal immune system and thereby initiate a cascade of events leading to ileal inflammation in SAMP1/Fc mice. A potential explanation comes from our recent demonstration that SAMP1/Fc mice have increased permeability of the ileal epithelium compared with control strains (34). This change in epithelial permeability of the ilea is manifest by 3 wk of age, well before any histologic or biochemical evidence of inflammation in SAMP1/Fc mice. By contrast, no such permeability changes are seen in the colon of SAMP1/Fc mice (34). These mice do not develop colitis, although, as shown, RELMβ mRNA was also increased in the colon. Other studies have also suggested that disruption of barrier function can predispose the intestine to chronic inflammatory processes (35, 36, 37). Thus, we propose a model whereby luminally secreted RELMβ, in the context of a compromised mucosal barrier, can activate cells of the innate and/or adaptive immune systems in the lamina propria leading to the observed chronic ileitis in these mice.

Our current demonstration that naive BM macrophages respond to exogenously added RELMβ by secreting TNF-{alpha}, IL-6, and RANTES suggests one possible mechanism whereby RELMβ may participate in the initiation and maintenance of the chronic ileitis that develops in the SAMP1/Fc mouse strain. Similar to TNF-{alpha}, IL-6 has been shown to play a key role in the pathogenesis of Crohn’s disease. Patients with Crohn’s disease have elevated serum and tissue levels of IL-6 (38, 39). Recent studies show that IL-6 is elevated in several models of colitis and in the SAMP1/Yit mouse model of ileitis (40, 41, 42). Blockade of IL-6 in a mouse model of IL-23-mediated colitis and a T cell-mediated colitis significantly attenuated inflammation (40, 41). RANTES has been shown to have a key role in the transition from acute to chronic disease in experimental models of colitis in addition to triggering leukocyte adhesion to the inflamed intestinal microvasculature (43, 44, 45). Thus, the ability of RELMβ to stimulate macrophages and perhaps other cells of the mucosal immune system, to secrete TNF-{alpha} and IL-6 as well as the chemokine, RANTES, suggests that RELMβ is a potential key mediator of the pathogenesis of terminal ileitis seen in the SAMP1/Fc mouse model of Crohn’s disease. To define the mechanisms whereby RELMβ may, either directly or indirectly, regulate the responses of the innate and adaptive immune system to initiate and/or exacerbate an inflammatory cascade will require further study.

In conclusion, our findings suggest that enhanced expression of RELMβ in the context of increased mucosal permeability has a role in the initial pathogenesis of inflammation in Crohn’s disease. The demonstrated ability of RELMβ to elicit TNF-{alpha}, IL-6, and RANTES production by activating BM-derived macrophages further supports the hypothesis that this goblet cell-specific peptide has a proinflammatory role in IBD pathogenesis. Our data also emphasize that goblet cells may play a critical role in the development of intestinal inflammation and the pathogenesis of IBD through their secretion of RELMβ.


   Acknowledgment
 
We thank Jenny Buzan for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 This work was supported by grants to S.M.C. (National Institutes of Health (NIH) R01 DK06475, P01 DK57880), F.C. (P01 DK57880, R01 DK42191, R01 DK55812), and G.D.W. (NIH R01 AI39368) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and through the Molecular Biology and Morphology Cores of the University of Virginia NIH/NIDDK Digestive Diseases Research Core Center Grant (DK50306) and University of Pennsylvania NIH/NIDDK Center Grants (DK56703). S.L.B. was also supported by an Institutional NIH Postdoctoral Fellowship (T32 DK07769) and by a UNCF-Merck Postdoctoral Science Research Fellowship. M.-L.W. was supported by NIH KO8 DK066206. Back

2 S.L.B. and A.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Steven M. Cohn, Digestive Health Center of Excellence, Box 800708, University of Virginia Health System, Charlottesville, VA 22908. E-mail address: SC6W{at}virginia.edu Back

4 Abbreviations used in this paper: Tff3, intestinal trefoil factor 3; RELMβ, resistin-like molecule β; FIZZ2, found in inflammatory zone 2; mFIZZ, mouse FIZZ; Muc2, mucin 2; SPF, specific pathogen-free; GF, germfree; IBD, inflammatory bowel disease; DSS, dextran sulfate sodium; PAS, periodic acid-Schiff; ARE, AU-rich element; BM, bone marrow. Back

Received for publication August 11, 2007. Accepted for publication August 29, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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