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. 1998 Jun;112(3):435–442. doi: 10.1046/j.1365-2249.1998.00595.x

Colonic explant production of IL-1 and its receptor antagonist is imbalanced in inflammatory bowel disease (IBD)

S Dionne 1, D D D'Agata 1, J Hiscott 1, T Vanounou 1, E G Seidman 1
PMCID: PMC1904987  PMID: 9649212

Abstract

IBD is associated with an increased activation of intestinal immune cells, which causes overproduction of proinflammatory cytokines such as IL-1β. IL-1β is implicated in mediating the sustained inflammatory response. IL-1 receptor antagonist (IL-1Ra), the naturally occurring inhibitor of IL-1, has been shown to have beneficial effects in experimental models of colitis. In this study we investigated the hypothesis that an imbalance between IL-1 and IL-1Ra exists in IBD by measuring their secretion by explant cultures of colonic biopsies. Freshly homogenized biopsies from involved tissue in IBD patients exhibited significantly lower IL-1Ra/IL-1β ratios than control and uninvolved IBD mucosal tissue. Using explant cultures, in vitro production of IL-1β and IL-1Ra increased progressively during the 4–18-h culture periods. IL-1β secretion was higher in supernatants from involved Crohn's disease (CD) and ulcerative colitis tissue compared with control tissue, and IL-1β levels increased with severity of inflammation. IL-1Ra secretion was not elevated in involved IBD samples, but significantly higher levels were released when moderate to severely involved tissue samples were compared with non-inflammatory controls. Similar to freshly homogenized tissue, explant studies showed that the IL-1Ra/IL-1β ratios were significantly decreased in involved IBD tissue, but not in uninvolved CD or inflammatory control specimens. These data support the hypothesis of an imbalance between IL-1β and IL-1Ra in IBD.

Keywords: IL-1, IL-1Ra, Crohn's disease, ulcerative colitis, cytokines

INTRODUCTION

Crohn's disease (CD) and ulcerative colitis (UC), entities collectively referred to as IBD, are characterized by chronic, relapsing intestinal inflammation. Although their aetiologies remain unknown, several lines of evidence support a role for the mucosal immune system in the initiation and perpetuation of these disorders [1–4]. An increased number and activation of mucosal lymphocytes, macrophages and polymorphonuclear cells are associated with IBD [1,5], which result in heightened production of proinflammatory cytokines including tumour necrosis factor (TNF), IL-1, IL-6, IL-8 and interferon-gamma (IFN-γ) [3,6–8]. The liberation of these inflammatory mediators affects epithelial [3,9] as well as other mucosal cell functions, and ultimately leads to tissue damage [1–4,10].

Counter-regulatory mechanisms normally exist that limit the deleterious effects of proinflammatory cytokines, including the inhibition of cytokine production [11–14], down-regulation of cytokine receptors on target cells [15], increased production of anti-inflammatory cytokines [16–18], and the production of inhibitors [19–21]. The latter mechanism has gained increased attention since soluble receptors have now been described for a large number of cytokines. These soluble peptides are usually generated by proteolytic cleavage of the extracellular domain of the receptor, antagonizing the binding of the ligand to the cellular receptors [21].

The proinflammatory activity of IL-1 is modulated by the IL-1 receptor antagonist (IL-1Ra), which binds IL-1 receptors, yet elicits no intracellular response [3,21,22]. IL-1Ra inhibits IL-1 biological activity, provided that the IL-1Ra/IL-1 ratio is sufficiently elevated. A 10–500-fold excess of IL-1Ra is necessary to block 50% of IL-1 responses, depending upon the target cell type [3,21–23]. The production of soluble receptors to IL-1 and IL-1Ra are thus key components of a protective mechanism to limit the deleterious effects of inflammatory mediators and most probably to assist in the resolution of the inflammatory process [15,20–23]. This hypothesis is supported by studies that document their increased levels in a number of inflammatory conditions, such as sepsis and rheumatoid arthritis (RA) [21–23].

A physiological protective role for IL-1Ra in IBD was elegantly demonstrated by Cominelli et al., who showed that the administration of recombinant IL-1Ra attenuated [24], whereas neutralization of IL-1Ra exacerbated and prolonged inflammation in a rabbit colitis model [25]. Similarly, the severity of colitis in the rat was inhibited by rhIL-1Ra [26]. Furthermore, recent studies point to a novel genetic association between the gene for IL-1Ra and UC [27]. Using quantitative polymerase chain reaction (PCR) methodology, we have recently reported an increase in IL-1 mRNA transcripts in colonic biopsies from paediatric IBD patients [28]. Collectively, these studies provide evidence supporting the hypothesis that proinflammatory IL-1 and an imbalance with its receptor antagonist play an important role in the pathogenesis and progression of IBD in humans. To investigate further this hypothesis, in vitro production of IL-1β and IL-1Ra by explant cultures of colonic mucosal biopsies from paediatric patients with IBD were measured.

PATIENTS AND METHODS

Material

Dulbecco's PBS, CMRL 1066 and penicillin–streptomycin were purchased from Gibco BRL (Life Technologies, Burlington, Ontario, Canada). Fetal calf serum (FCS), Tris, D-retinol acetate were from Sigma (St Louis, MO). Anti-proteases AEBSF, leupeptin, aprotinin and pepstatin were obtained from ICN (Mississauga, Ontario, Canada). Antipain was from Boehringer (Laval, Quebec, Canada), gentamicin from Schering (Pointe-Claire, Quebec, Canada) and amphotericin B from Bristol-Meyers Squibb (Montreal, Quebec, Canada). Immunoassays were ordered from R&D Systems (Minneapolis, MN).

Organ cultures

Rectal biopsies were obtained from consenting patients at the time of colonoscopy for evaluation of gastrointestinal symptoms. Six biopsies were taken from macroscopically diseased areas of the rectal mucosa of IBD and inflammatory control patients. If no macroscopic lesions were visible, six specimens were taken from the rectum. To determine mucosal disease severity, one representative sample was fixed in formalin for routine histopathology. An experienced pathologist blindly classified patients into one of the four groups ranging from normal histology (score 0) to severe inflammation (score 3). All patients were followed clinically for at least 6 months in order to correlate tissue cytokine levels with clinical course (early relapsers). The study protocol and consent forms were approved by the Ethics Review Committee of Ste-Justine Hospital.

Colonic biopsies were immediately transported to the lab in CMRL medium on ice, weighed, and either homogenized in 1 ml ice-cold PBS pH 7.4 containing an anti-protease cocktail of AEBSF (2.5 mg/ml), leupeptin and aprotinin (0.75 mg/ml) and antipain and pepstatin (0.5 mg/ml), or immediately placed in organ culture dishes.

Biopsies were placed mucosal surface up on a sterilized steel grid in an organ culture dish (Falcon). The outer well was filled with 0.15 m NaCl. The central well received 1 ml of medium, consisting of CMRL 1066 supplemented with 10% heat-inactivated FCS, Tris 20 mm, 100 U penicillin–streptomycin, 50 μg gentamicin, 0.25 μg amphotericin B and 1 μg β-retinyl acetate. Cultures were maintained for either 4 h (group A) or 18 h (group B) at 37°C in an humidified 5% CO2/95% O2 atmosphere, and gently rocked at 20 cycles/min. At the end of each culture period, the supernatant was collected, aliquoted and stored at −70°C, while biopsies were either homogenized (as above) or frozen in liquid nitrogen.

Patient populations

Overall, rectal biopsies were obtained from 52 paediatric patients with CD, 20 with UC, 13 from patients with other forms of colitis (inflammatory controls), and from 42 non-inflammatory controls. Patients from each patient group were classified according to whether their rectal biopsies were cultured for 4 h or 18 h (groups A and B, respectively). The clinical characteristics of the patient population groups studied are presented in Table 1.

Table 1.

Clinical characteristics of the patient groups, classified by duration of colonic explant cultures

graphic file with name cei0112-0435-t1.jpg

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Group A (4 h cultures)

Among the 13 CD patients with rectal inflammation (involved CD) in group A, eight were newly diagnosed, while five others had been previously diagnosed (1–2.5 years). Eight had colonic CD, three ileocolitis, and two gastroileocolitis. Eight patients were classified as Crohn's pancolitis, two each had left-sided colitis and proctitis, and one multifocal colitis. All eight newly diagnosed patients were untreated, as were two other cases. Among the three remaining patients, one was on steroids, 5-ASA and cyclosporin, and the two others were receiving steroids and azathioprin. Two of these three patients were in the moderately to severely inflamed group. Therefore all but one patient in the mildly inflamed group were untreated at the time of biopsy.

Of the 11 CD patients in group A whose rectal biopsies were uninvolved, five were newly diagnosed, while the six others had disease duration from 0.5 to 7 years and were on treatment. Two patients were in remission, the others had ileocolitis (n = 4), colitis (n = 2), duodenoileitis (n = 1) and ileitis (n = 2). Of the six patients with colonic involvement, two presented with endoscopic features of early CD and one had a pancolitis, with rectal sparing. The three others had focal colitis mainly localized to the transverse colon.

Of the 12 UC patients, six were newly diagnosed. Disease duration among the others ranged between 4 months and 9 years. All UC patients' rectal biopsies were involved. Disease distribution was pancolonic in nine, and left-sided colitis in three. Two patients were on topical steroid enemas (with or without oral 5-ASA), one each were receiving steroids and 5-ASA and oral 5-ASA only, and one other sulfasalazine.

The inflammatory control (IC) group comprised seven patients with acute, self-limited colitis. The acute self-limited colitis patients consisted of antibiotic associated colitis (n = 3), and culture-negative acute colitis (n = 3). The other had positive culture for Blastocytis hominis and Endolimax nana.

The non-inflammatory control (N-IC) group comprised three patients with lactose intolerance, 14 with recurrent abdominal pain, seven cases investigated for familial polyposis, one ganglioneuroma, one recurrent rectal prolapse, and four patients with an irritable bowel.

Group B (18 h cultures)

Eight of the 14 CD patients with rectal involvement in this group were newly diagnosed. Twelve had ileocolitis (three with gastroduodenal extension), and one each had colitis and proctitis. Seven were classified as pancolitis, two each as hemicolitis, left-sided colitis and proctitis, and one had multifocal colitis. Ten patients were untreated and one was on steroids, 6-MP and 5-ASA. The others received steroids and/or 5-ASA (Table 1).

The 14 CD patients in group B with uninvolved rectal mucosa had ileitis (n = 4), ileocolitis (n = 4), colitis (n = 4), oesophagogastritis (n = 1) and oesophagogastroileitis (n = 1). Of the eight patients with colonic involvement, four had focal colitis and two pancolitis and hemicolitis with rectum spared. Among the 11 patients who were newly diagnosed, six had colonic inflammation.

Five of the nine UC patients were newly diagnosed. Seven patients had pancolitis, one had hemicolitis and one proctitis. The IC group consisted of six cases of acute, self-limited colitis. Three had antibiotic associated colitis and three culture-negative colitis. Three had pancolitis, two proctitis and one focal colitis. The N-IC group comprised three cases with lactose intolerance, two recurrent abdominal pain, two cases investigated for familial polyposis, one for vascular abnormalities, and five patients with an irritable bowel.

Cytokine determinations

Cytokine assays were performed by commercially available high sensitivity and specific ELISA kits, according to the manufacturers' instructions: IL-1β (Quantikine HS, detection limit 0.05 pg/ml); IL-1Ra (Quantikine, detection limit 6.5 pg/ml). The antibody used for IL-1β determination recognizes mature IL-1β, with cross-reactivity to proIL-1β of 13%. There were negligible amounts of cytokine in the FCS utilized (< 0.05 pg/ml). FCS did not affect cytokine determinations, but was essential for explant viability for the 18 h cultures.

Statistical analysis

Non-normally distributed data were expressed as medians. The variation between group medians was tested using the Kruskal–Wallis non-parametric test. Thereafter, the differences between groups were assessed with the two-tailed Mann–Whitney U-test. The effect of clinical variables on cytokine excretion within each group was tested with one-tailed Mann–Whitney U-test or Spearman rank correlation coefficient test. P < 0.05 was considered statistically significant.

RESULTS

Tissue cytokine concentration

Preliminary studies were performed in freshly homogenized mucosal samples (Table 2). Low levels of IL-1β were detected in the N-IC group (0.20 pg/mg). The mean values observed in uninvolved CD and IC were about two-to-three-fold higher (0.39 and 0.57 pg/mg, respectively), whereas in involved IBD tissue (nine CD, four UC) it was significantly (nine-fold) higher (1.80 pg/mg, P < 0.005). IL-1Ra concentration was not different in the two IBD groups (85 and 159 pg/mg) compared with the N-IC (123 pg/mg). Consequently, the IL-1Ra/IL-1β ratio was significantly decreased in mucosal tissue from involved IBD (P < 0.01), as shown in Table 2.

Table 2.

Cytokine levels in freshly homogenized colonic biopsies

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The tissue cytokine concentration in mucosal biopsies was also measured after explant culture for either 4 h or 18 h. Overall, compared with freshly homogenized tissue, IL-1β content in cultured explants was 10-fold higher (1.9), whereas IL-1Ra was relatively unchanged (164 pg/mg) in cultured N-IC biopsies. In uninvolved CD biopsies, the corresponding levels were 2.3 and 253 pg/mg, while involved IBD tissue levels were 4.5 pg/mg for IL-1 and 171 pg/mg for IL-1Ra. When the ratio of IL-1Ra to IL-1β was calculated for cultured biopsies, a significantly decreased ratio (P < 0.05) was again observed for involved IBD mucosa (26), compared with N-IC (123) and uninvolved CD (184).

IL-1β and IL-1Ra secretion

Group A. IL-1β secretion (Fig. 1) by involved CD mucosa after 4 h of culture was markedly increased compared with N-IC (0.48 versus 0.07 pg/mg tissue, P < 0.01). Elevated IL-1β concentrations were also observed in involved UC medium (0.40 pg/mg, P < 0.05). CD and UC specimens with moderate/severe inflammation released more IL-1β (P < 0.05) compared with mildly inflamed CD and UC tissue (Table 3).

Fig. 1.

Fig. 1

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Box-and-whisker plot showing IL-1β secretion by explant cultures of colonic mucosal biopsies during a 4-h culture period. Results are expressed in pg/mg tissue. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. *P < 0.05; **P < 0.01.

Table 3.

Comparison between the tissue inflammation severity and cytokine secretion in colonic Crohn's disease (CD) and ulcerative colitis (UC)

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Comparable amounts of IL-1Ra (Fig. 2) were secreted by cultured CD and UC explants (20.0 and 25.7 pg/ml) compared with biopsies of all other groups (median 17.7–25.0 pg/ml). As for IL-1β, higher IL-1Ra release was noted for more severely inflamed mucosal specimens (Table 3).

Fig. 2.

Fig. 2

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Box-and-whisker plot showing IL-1Ra secretion by explant cultures of colonic mucosal biopsies during a 4-h culture period. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. Results are expressed in pg/mg tissue.

The IL-1Ra/IL-1β ratio was decreased in involved CD and UC compared with controls (39 and 81 versus 166; P < 0.01 and P < 0.05, respectively), but not in uninvolved CD and IC (235 and 193, Fig. 3). The IL-1Ra/IL-1β ratio was not significantly different for more severely inflamed CD mucosa compared with mildly involved mucosal specimens (Table 3).

Fig. 3.

Fig. 3

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IL-1Ra/IL-1β ratio measured in culture supernatants. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. *P < 0.05; **P < 0.01.

Group B. After 18 h of culture, IL-1β secretion was greatly increased in UC mucosal explants compared with N-IC (0.92 versus 0.05 pg/mg; P < 0.01). Significant increases were also observed for the involved CD and IC groups (Fig. 4). Paralleling IL-1β, IL-1Ra secretion was also significantly higher in UC mucosa compared with N-IC (50.0 versus 11.4 pg/mg). However, IL-1Ra release by CD and IC mucosa explants was not different (Fig. 5) compared with the N-IC group. The IL-1Ra/IL-1β ratio was significantly decreased in involved CD and UC compared with controls at 18 h (42 and 43 versus 184, P < 0.01). The uninvolved CD mucosal explants secreted IL-1β and IL-1Ra in proportions similar to N-IC (Fig. 6). The IL-1Ra/IL-1β ratios for the IC were highly variable, ranging from 20 to 740, but the median (96) was not different from that of N-IC.

Fig. 4.

Fig. 4

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IL-1β secretion by explant cultures of colonic mucosal biopsies during an 18-h culture period. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. *P < 0.05; **P < 0.01.

Fig. 5.

Fig. 5

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IL-1Ra secretion by explant cultures of colonic mucosal biopsies during an 18-h culture period. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. **P < 0.01.

Fig. 6.

Fig. 6

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IL-1Ra/IL-1β ratio measured in culture supernatants. CDinv, Crohn's disease tissue, histologically involved; CDun, Crohn's disease, uninflamed; UC, ulcerative colitis; IC, inflammatory controls; N-IC, non-inflammatory controls. *P < 0.01.

IL-1β secretion was not increased by prolonging the culture time in most groups, while that of IL-1Ra generally decreased. The UC specimens were an exception, with a two-to-three-fold increase observed. Although this could be a specific characteristic of UC mucosa, a difference in the UC patient between group A and B is a more likely explanation. As shown in Table 1, UC specimens in group B were more severely inflamed than those in group A. In addition, a smaller proportion of patients in group B was treated. Nevertheless, the IL-1Ra/IL-1β ratios were similar in the two groups.

In a group of 16 biopsies, cytokine content was measured in freshly obtained paired biopsies and compared with values after culture. Total IL-1β increased to reach 720% during explant culture, while IL-1Ra levels were 147% of those detected in uncultured biopsies.

Influence of clinical and histopathological parameters on cytokine secretion

Cytokine secretion by involved CD and UC biopsies correlated to the inflammation score severity (Table 3), but not to the type of infiltrating cells (chronic, predominantly acute or mixed). Cytokine release was not related to age, sex or disease location. Although our data did not reveal an effect of treatment, the limited number of samples did not allow us to negate this possibility.

All patients were followed for a minimum of 6 months and up to 3 years. Patients presenting with a clinical relapse in the 6 months following the time when the analysed biopsies were obtained were considered early relapsers. The percentage of relapsers ranged from 25% to 46% in group A, but was 77% for involved CD in group B. Early relapsers from uninvolved CD in group A released significantly more IL-1β (0.22 versus 0.07 pg/mg, P < 0.05). Uninvolved CD relapsers' IL-1Ra release was not significantly less than in patients who remained in remission after 6 months (16.5 versus 25.0 pg/mg and 8.5 versus 28.9 pg/mg in groups A and B, respectively; NS). The lack of a statistically significant difference may be due to the limited number of patients in each group.

DISCUSSION

IL-1 has been implicated as one of the key proinflammatory cytokines involved in the pathogenesis of the chronic inflammation and mucosal injury in IBD [1–4,6–8,24–27]. Previous studies have documented increased mucosal IL-1 expression and production in both CD and UC [3,6–8]. The activity of the agonists cell-associated IL-1 and secreted IL-1β is modulated by two membrane-bound IL-1 receptors (IL-1RI and IL-1RII), and by four specific peptides, the soluble forms of IL-1RI and IL-1RII and two forms of IL-1Ra [21–23]. While monocytes and neutrophils produce a 22–26-kD secreted peptide (sIL-1Ra), epithelial cells synthesize a 16–18-kD peptide lacking the signal sequence that remains intracellular (icIL-1Ra) [21,23,29]. Both transcripts are expressed in rabbit intestine [30], and following induction of colitis colonic IL-1Ra production is delayed with respect to IL-1 production [25].

In the present study we assessed the balance between IL-1 and IL-1Ra in IBD by measuring their secretion by cultured explants of colonic biopsies. The reliability of this in vitro model has been established for measuring IL-1 [31,32], and other cytokines [33–35] in IBD. IL-1 tissue content has been demonstrated to be greatly increased during culture, and IBD mucosa secretes significantly higher amounts of IL-1 [31,32], as observed in the present study. Furthermore, our results show for the first time that tissue IL-1Ra content also increased during culture, and that substantial amounts of IL-1Ra are secreted in vitro. Organ cultures are not pyrogen-free, and it is possible that lipopolysaccharide (LPS) or other bacterial products modulated cytokine production in vitro. Furthermore, we cannot exclude an effect of factors present in FCS, or the possibility that other cytokines liberated in vitro influenced IL-1β and IL-1Ra release.

Four studies to date have reported measurements of IL-1Ra in IBD mucosal tissue [36–39]. Although absolute values differed substantially, a decreased IL-1Ra/IL-1 ratio in involved IBD tissue was a common feature. However, incomplete or contradictory results were presented for the IL-1Ra/IL-1 ratio in uninvolved IBD as well as in non-IBD inflammatory control tissue. Validation of the theory regarding a putative imbalance between IL-1 and IL-1Ra production as a primary defect in IBD required assessment of these two important controls. The IL-1Ra/IL-1β ratio in IBD uninvolved tissue was higher than that in involved mucosa, but lower than control mucosa in two studies [38,39]. In the one other report, uninvolved CD values were comparable to controls but increased compared with uninvolved UC [36]. There was a trend toward lower IL-1Ra/IL-1 ratios in uninvolved CD compared with uninvolved UC in two studies [36,39]. We did not observe any alteration in the IL-1Ra/IL-1β ratio in uninvolved CD tissue. The proportion of both cytokines secreted was comparable to that of N-IC.

Although a low IL-1Ra/IL-1β ratio seems to be a specific characteristic of involved IBD tissue, extensive studies on non-IBD colitis have not been conducted. An IL-1Ra/IL-1 ratio comparable to controls was found in non-IBD colitis in two studies [36,37], while it was comparable to involved IBD values in another [39]. Hyams et al. [38] also observed a lower ratio compared with control biopsies in their four non-IBD inflammatory control specimens. No differences were found in IL-1Ra/IL-l ratio between IC and N-IC tissue.

We noted some variability in the IL-1Ra/IL-1 ratio between IC samples. Several factors could have contributed to this. Differences in inflammation severity may play a role in the release of IL-1Ra and IL-1, but as stated, most IC specimens had mild inflammation. Different infectious agents may cause divergent cytokine responses. However, comparison of the results in antibiotic-associated colitis revealed as much variability as that observed with the other IC samples. The timing of tissue sampling with respect to onset of the infection may have influenced the IL-1Ra and IL-1 levels. In a model of mycobacterial infection, IL-1Ra production was highest early on, when monokines were dominating. After several days, a second peak of IL-1 occurred without a concomitant increase in IL-1Ra [40]. Analysis of the effect of the type of inflammatory cell infiltrate on the IL-1Ra/IL-1β ratio has not previously been examined. The IC group tissue generally had mild, predominantly acute inflammatory cell (neutrophilic or eosinophilic) infiltrates. Among the CD group, the specimens having the higher IL-1Ra/IL-1β ratios also had acute inflammatory cell infiltrates. Our data suggest that the type and severity of inflammatory cell infiltrates influence the IL-1Ra/IL-1β ratio, whether in IC or IBD tissue. Our data from a group of eosinophilic colitis patients provide additional evidence supporting this observation. After 4 h of culture, the IL-1Ra/IL-1 ratio in the supernatants for the latter group (n = 7) was maintained in the same range noted for N-IC tissue (median 151). The IL-1Ra/IL-1β ratios were low in IBD tissue with mixed (acute and chronic) or those with primarily chronic inflammation, as had been found in studies using lamina propria mononuclear cells isolated from involved IBD mucosa [37,41]. However, the ratio was uniformly lower in the latter studies, probably because epithelial cells, important sources of IL-1Ra, were absent [37].

The experimental model used in this study may be physiologically more relevant than measurement of cytokines in isolated cells or homogenized tissue, since the products secreted in the extracellular compartment and available to adjacent cells are also measured. The pathophysiological significance of secreted IL-1β and IL-1Ra is quite obvious. Homogenates contain IL-1α and inactive proIL-1β as well as icIL-1Ra. The role of the latter peptides remains elusive. ProIL-1β is the inactive precursor of IL-1β and is not secreted [23,42]. In monocytes, 90% of total IL-1β consists of inactive proIL-1β [42]. This reservoir of proIL-1β may be released upon cell injury or apoptosis and subsequently cleaved by extracellular converting enzymes [42]. IL-1α and IL-1α precursors remain in the cell, but are potentially active. IL-1α could also be released after cell death, and the possibility of an intracellular function has been suggested [29]. A physiological role for icIL-1Ra has not yet been established, but it is released along with IL-1α and proIL-1β subsequent to cell death. An intracellular role has also been suggested, and icIL-1Ra may antagonize cytosolic IL-1α by binding to nuclear membrane IL-1 receptors [29]. Anti-IL-1Ra recognizes both forms of IL-1Ra, while antibodies directed against mature IL-1β cross-react to different degrees with proIL-1β [42].

The relationship between IL-1Ra/IL-1 ratio fluctuations and the relapsing-remitting course of IBD remains elusive, as does the effect of these changes on the evolution of the disease. Miller et al. [43] reported that elevated IL-1Ra and IL-1Ra/IL-1 ratios are correlated with a more rapid resolution of attacks of arthritis in Lyme disease. Median IL-1Ra/IL-1 ratios in our study were about two-fold lower in uninvolved tissue of CD patients who relapsed early, in keeping with this theory. Cytokines produced by intestinal cells can modulate IL-1 and IL-1Ra liberation [29,44], and the composition of mucosal inflammatory infiltrates may have a key role in this modulation. IL-1Ra may be induced by a number of cytokines, including IL-1, TNF-α, transforming growth factor-beta (TGF-β), IL-4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) [23,29,44].

Our results show that the type and severity of mucosal inflammation is a major determinant of IL-1Ra secretion, rather than disease location. IBD mucosa is the site of dense accumulation of recently emigrated CD14+ L1+ macrophages [45]. IL-1β was found to be secreted mostly by immature macrophages which are increased in involved and non-involved IBD mucosa, and particularly in involved UC tissue [46]. The IL-1Ra/IL-1 ratio increases during macrophage differentiation, as mature macrophages constitutively produce IL-1Ra, but do not respond to LPS or IgG [47]. It is thus tempting to speculate that these immature, newly recruited macrophages may be responsible for the decreased IL-1Ra/IL-1 ratio we observed in involved IBD mucosa.

Acknowledgments

Research Fellowship support was awarded from the Canadian Association of Gastroenterology/Astra Pharma Inc./MRC-PMAC Program (S.D., I.D.D.). This study was supported in part by a research grant (E.G.S., J.H.), and a summer student award (T.V.) from the Crohn's and Colitis Foundation of Canada. Research Scholarship Awards (E.G.S., J.H.) were obtained from the Fonds de Recherche en Santé du Québec (FRSQ). Technical equipment purchase was funded by the Ste-Justine Hospital Foundation Rainbow of Fashion Research Fund. The authors wish to thank Anne Duhaime and Denise Lévesque for their valuable technical assistance, and Marie-José Claveau for expert secretarial assistance. We are extremely grateful to the members of our Division of Pediatric Gastroenterology for kindly supplying the biopsy specimens.

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