The therapeutic experience of IL-10 in animal models of colitis is very encouraging. In vitro studies have shown that exogenous IL-10 can down-regulate the enhanced pro-inflammatory cytokine release from lamina propria mononuclear cells isolated from patients with Crohn’s disease. Early clinical studies in untreated, as well as steroid-refractory Crohn’s disease patients, suggested that IL-10 might have strong potential in the treatment of human diseases. Colombel et al[16] performed a double blind controlled trial to evaluate the safety and tolerance of recombinant human interleukin 10 (IL-10, Tenovil) in subjects operated on for Crohn’s disease. Tenovil treatment for 12 consecutive weeks in patients with Crohn’s disease after intestinal resection was safe and well tolerated. No evidence of prevention of endoscopic recurrence of Crohn’s disease by Tenovil was observed.

Unfortunately, this enthusiasm was dampened by the results of 2 multinational multicenter studies reporting the efficacy and safety of daily subcutaneous injections of recombinant human IL-10 (rhuIL-10) in patients with moderately active and steroid-refractory Crohn’s disease[17,18]. Fedorak et al [17] reported a 24-week double-blind placebo controlled study of 95 patients with moderately active Crohn’s disease randomized to receive subcutaneous rhuIL-10 at 1 of 4 doses (rhuIL-10:1, 5, 10, or 20 µg/kg) or placebo daily. After a 28-day treatment period, a modest response was seen in the group receiving the 5 µg/kg dose, whereby 23.5% of patients (n = 17) showed improvement compared with 0% of patients treated with placebo (n = 23). Interestingly, higher doses were less effective, although there was no difference in reported adverse events between doses. During the course of a 20-week follow-up period, disease recurrence requiring therapeutic intervention occurred in 70% of placebo-treated patients compared to 55% of the rhuIL-10-treated group[17].

In the study by Schreiber et al[18] involving 329 therapy refractory patients with active Crohn’s disease, no significant differences in the induction of clinical remission were seen between placebo and rhuIL-10 at any dose (1, 4, 8, and 20 µg/kg), although clinical improvement was achieved in 46% of patients in the 8 µg/kg group compared with 27% of patients receiving placebo (P = 0.03). The authors also determined the nuclear levels of transcription factor NF-κB p65 and cytoplasmic concentrations of its inhibitor IκBα in ileocolonic biopsy specimens. They noticed significant differences between patients who responded to rhuIL-10 treatment and those who did not, implicating the inhibition of NF-κB nuclear translocation as one possible mechanism of the action of this therapy[18].

Recent studies linking high systemic concentrations of IL-10 with headache, fever[19], and anemia[20] have further aroused the enthusiasm for IL-10 as a therapeutic drug in treatment of IBD. Indeed, the biology of IL-10 is complex. In addition to its effects in down-regulating pro-inflammatory events, IL-10 has been shown to exert immunostimulatory effects by up-regulating MHC II expression on B lymphocytes and inducing cytotoxic T-cell differentiation. Clinical data support these immunostimulatory effects of IL-10 at high systemic doses. Tilg et al[19] reported that patients with Crohn’s disease receiving rhIL-10 at high doses experienced a significant increase in serum neopterin and phytohaemagglutin (PHA) induced IFN-gamma production from whole blood cells. This phenomenon may be responsible for the lack of efficacy of high doses of IL-10 in the treatment of Crohn’s disease[19]. In fact, the emerging picture regarding the in vivo effects of IL-10 is one of both the anti- and pro-inflammatory effects, depending on the local concentrations of IL-10 achieved, the types of antigens present in the microenvironment, and the activation state of the immune cells in the vicinity. It is possible that high systemic doses of IL-10 alter the balance between its immunoregulatory and immunostimulatory effects, which would explain the bell-shaped curve in therapeutic efficacy reported in the clinical trials. In addition, it is entirely possible that the pharmacodynamics of daily systemic IL-10 administration does not allow for efficient delivery of the cytokine to the local sites of inflammation. The serum half-life of IL-10 is between 1.1 and 2.6 hours, thus, the cytokine may be cleared before reaching its target.

IBD in humans responds to high dose of intravenous interleukin 10, but this cannot be administered orally because stomach acid destroys it. Recently, studies have been published that looked at alternative methods of delivery of IL-10. The first study used the genetically engineered bacterium, Lactococcus lactis (L. lactis), as a delivery vehicle for IL-10. Steidler et al[21] hypothesised that an oral delivery system that effectively bypassed the stomach might provide a treatment which would cut the dose of IL-10 required and also reduce the risk of systemic side effects. Steidler and his colleagues[21] have engineered a non-pathogenic bacterium that produces interleukin 10, an anti-inflammatory cytokine that reduces inflammatory colitis. They then established two different mice models of inflammatory bowel disease. In the first model chronic colitis was induced by feeding dextran sulphate sodium to the mice over several weeks. When the recombinant bacteria were introduced into the mice stomachs, they survived the acid environment and released interleukin 10 in the colon. The second mouse model had its interleukin 10 gene knocked out. The resultant mice were shown to develop IBD spontaneously within 8 weeks of life. When the team administered the genetically engineered L. lactis to these mice at 3 weeks of age, colitis did not occur in the first place. They showed that daily intragastric administration of IL-10-secreting L. lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. High serum concentrations of interleukin 10 were not found, despite good uptake by inflamed cells of the gut mucosa. This approach may lead to better methods for cost-effective and long-term management of IBD in humans. Because the genetically engineered bacterium survives stomach acid, the cytokine can be delivered directly to the inflammatory target in the colon. This could reduce the dose needed and any systemic effects.

However, the release of such genetically modified organisms through clinical use raises safety concerns. Steidler et al[22] replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene to address this problem. This thyA-hIL10⁺L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium’s capacity of secreting hIL-10 were validated in vivo in pigs. This approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strains acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

Two technical hitches must be ironed out, however, before human trials can be considered. Human interleukin 10 is slightly different from murine interleukin 10, so the L. lactis has to be re-engineered. The second obstacle is that human bile is stronger than mouse bile and is likely to kill the bacteria as they pass through the stomach and duodenum[23].

IL-10 is an anti-inflammatory cytokine that suppresses the T helper 1 immune response and down-regulates macrophages and monocytes. The therapeutic effect of systemic administration of IL-10 for patients with IBD, however, has not been satisfactory. Several studies have indicated that active monocytes, such as macrophages and T cells, play an important role in the pathogenesis of chronic human IBD, although the etiology remains unclear. Manipulation of these cells appears essential for the treatment of patients with IBD. Recently, considerable attention has been paid to the use of polymer microspheres for the sustained release of various drugs and the targeting of therapeutic agents to their sites of action.

Nakase et al[24,25] developed gelatine microspheres (GM) containing IL-10 (GM-IL-10), which can be released sustainedly to a local site without losing bioactivity. They administered these microspheres to IL-10 knocked out mice rectally to investigate whether this treatment could ameliorate colitis. Colonic inflammation in mice treated with GM-IL-10 was remarkably reduced compared to those treated with IL-10 alone. Macroscopic and microscopic examination revealed marked improvement of colitis in IL-10(-/-) mice treated with GM-IL-10. mRNA expression of IL-12 in Mac-1-positive cells in GM-IL-10-treated mice was significantly decreased compared with that in the mice treated with IL-10 alone. CD40 expression in Mac-1-positive cells in GM-IL-10-treated mice was decreased more prominently than that in mice treated with IL-10 alone. The therapeutic effects of GM-IL-10 were associated with decreased expression of IL-12 mRNA and down-regulation of CD40 expression in Mac-1-positive cells. Additionally, intestinal administration of these microspheres significantly improved colitis with a decrease in histological score, myeloperoxidase activity, and nitric oxide production compared with those treated with free agents. Gene expressions of TNF-α, IL-1β, and IFN-γ were down-regulated in treated animals. Serum IL-10 levels and systemic macrophages were unchanged after treatment[26].

These data suggest that local macrophages in the intestine play a critical role in the initiation of chronic colitis in the animal model of IBD. A drug delivery system using these microspheres containing immunomodulatory IL-10 (GM-IL-10) might be useful for treatment of patients with IBD.

IL-10 is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. It may be possible to use replication-deficient adenoviral vectors to deliver the IL-10 gene directly to gastrointestinal epithelial cells. This would lead to localized high level IL-10 release for a short duration that is determined by the lifetime of the infected cells. IL-10 adenoviral gene therapy has proved very successful in murine models of rheumatoid arthritis[27], a condition with many immunological similarities to Crohn’s disease. Previous studies have demonstrated that adenoviral vectors, when delivered by rectal infusion, could infect intestinal epithelial cells[28]. This approach may be limited by the host anti-adenoviral immune response, that has limited gene expression and prevented re-treatment with other adenoviral vectors[29]. However, there is evidence that the delivery of immunoregulatory genes, such as IL-10, would diminish both the cell-mediated and humoral anti-adenoviral responses[30,31].

Barbara et al[32] reported that gene transfer was achieved by intraperitoneal injection of non-replicating human type 5 adenovirus bearing IL-10 gene, either 24 hours before or one hour after intrarectal administration of dinitrobenzene sulphonic acid in rats. Colonic damage and inflammation were assessed macroscopically and by measuring the myeloperoxidase activity and leukotriene B4 concentrations. Gene transfer increased IL-10 protein in serum for up to six days. IL-10 gene transfer prior to colitis improved colitis macroscopically and histologically, and significantly reduced colonic myeloperoxidase activity and leukotriene B4 concentrations. In contrast, IL-10 gene transfer after the onset of colitis had no beneficial effect. Lindsay et al[33] showed that local adenoviral vectors encoding IL-10 (AdvmuIL-10) reverse colitis in IL-10-/- mice without systemic effects seen after intravenous administration.

Enhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of IBD. Sasaki et al[34] reported transfection of the IL-10 vector into endothelial cell cultures significantly reduced TNF-α induction, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46% +/- 6.6%) of the MAdCAM-1 induced by TNF-α (set as 100%) in non-transfected (control) cells.

These results suggest that gene therapy strategies using plasmid IL-10 vectors or an adenovirus-IL-10 construct may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn’s disease.

T cells, and in particular, regulatory T (Treg) cells, play a pivotal role in the control of intestinal inflammation[35,36]. Regulatory cells lacking anti-inflammatory cytokine IL-10 were unable to inhibit IBD, showing that IL-10 was required for the protective effects of lymphocytes in this setting[37].

van Montfrans et al[38,39] presented a novel method of IL-10 delivery to intestinal mucosal tissue by the use of transduced T cells. In these studies, the authors demonstrated that T lymphocytes could be engineered by retrovirus construct transduction to express high levels of IL-10 upon activation. With the goal of providing long-term therapy for Crohn’s disease, peripheral blood mononuclear cells were obtained from healthy adults and transduced with a retroviral vector containing IL-10 and green fluorescent protein (GFP). These CD4⁺ T lymphocytes responded to CD3/CD28 stimulation with a 6-fold increase in IL-10 production that was shown to be biologically active. Transduced cells had high expressions of the mucosal integrin α4β7, and displayed efficient binding to MAdCAM-1 expressing cells in vivo, suggesting that they would home to gut mucosa. Importantly, albeit in vitro, cells remained stably transfected for up to 4 months. In the classic CD45RB^high model, these IL-10 transduced CD4⁺ cells were able to effectively prevent the development of colitis, even when given up 14 days after the transfer of the CD45RB^high cells. Although circulating levels of IL-10 were not detectable, IL-10-GFP encoding messenger RNA was detected in colons of mice 15 weeks after cell transfer, and IL-10 was detected in the intestinal draining lymph nodes, spleen, and colon, indicating that the IL-10-GFP transduced CD4⁺ cells persisted in vivo and migrated into the large intestine. The mice receiving IL-10 transduced CD4⁺ cells exhibited a decrease in the production of TNF-α in the colon and a decreased production of IFN-γ and/or TNF-α in the intestinal draining lymph nodes. These results indicate that the transferred CD4⁺ cells are stimulated in vivo and therapeutically effective.

One of the advantages of using T cells as delivery vehicles is the likelihood that the cytokine of interest, in this case, IL-10, would primarily be released only on activation in local sites of inflammation, which would avoid the side effects known to be associated with high systemic levels and may further induce the development of immune-suppressor Treg cells in the area of inflammation. Secondly, the use of ex vivo transduction methodology would prevent systemic exposure to the retroviral vector, but still ensure sustained gene expression. However, the question remains, will even local delivery of IL-10 be effective in patients with IBD IL-10 certainly exhibits potent inhibitory effect on the activation and effector functions of T cells, monocytes, and dendritic cells, all of which are key players in intestinal inflammation. However, the colon from a patient with Crohn’s disease already secreted higher levels of IL-10 compared with control colons[40] and mononuclear cells isolated from the ileum of patients with Crohn’s disease appeared to be nonresponsive to IL-10[41]. These findings suggest that even if IL-10 is delivered directly to the intestinal mucosa, it may not be therapeutic in Crohn’s disease. This is mirrored by effects both in the IL-10 gene-deficient mouse chronic colitis model and in the dinitrobenzene sulphonic acute model of colitis[42] where IL-10 is able to prevent the development of colitis if given before inflammation develops, but is ineffective if given either exogenously or via gene transfer, once inflammation has become established. However, although IL-10 therapy may be ineffectual in treating active diseases, there may still exist a role for such therapy as maintenance treatment in Crohn’s disease. Low ileal IL-10 concentrations have been shown to predict relapse after ileocecal resection[43]. In addition, it may be that only certain subgroups of Crohn’s disease patients respond to IL-10 therapy, whether it is delivered systemically or locally. It is clear that even among patients with similar clinical presentations, there exists a large diversity of patterns of immune responses to environmental and bacterial antigens[44]. It is not known how patients with these types of immune reactivities respond to different therapies.