Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal with two main entities, Crohn’s Disease (CD) and Ulcerative Colitis (UC). The primary therapeutic goals are improvement of quality of life through induction and maintenance of remission, prediction, prevention, and treatment of complications, restoration of nutritional deficits, and alteration of the natural history of the disease [
Recombinant human Granulocyte-Macrophage Colony Stimulating Factor [GM-CSF (rHu GM-CSF)] [molgramostim (Mielogen)] is a human protein produced by a strain of Escherichia coli containing a plasmid encoding the human GM-CSF gene [
Lenograstim is the glycosylated recombinant form of human granulocyte colony stimulating factor with actions similar to those of molgramostim. Lenograstim is a valuable adjunct able to minimise the haematological toxicity of myelosuppressive chemotherapy in patients with malignant disease [
Of the currently in use animal models, the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced colitis, resembles human IBD in its various histological features, including infiltration of colonic mucosa by neutrophils and macrophages and increased production of inflammatory mediators [
Finally, malondialdehyde (MDA), is one of the final products of lipid peroxidation in the cells. An increase in free radicals during an inflammatory process as in colitis, causes overproduction of MDA. As a result, MDA level is commonly used as a marker of oxidative stress.
The aim of this study was to investigate the influence of the two growth factors; Granulocyte-Colony Stimulating Factor (G-CSF,
Sixty-two male Wistar rats of body weight greater than 250
Distal colitis was induced by intracolonic installation of 25
Animals were divided into 9 groups. The first two groups consisted of ten rats each (control groups, 20 animals in total). All other groups consisted of 6 rats each (42 animals in total). The number of animals used per group was based on a similar estimate to the ones that other relevant studies reported to have used across the literature. Those numbers were the highest possible for the available budget to allow safe conclusions. In other words, we recruited the highest possible number of mice for the given budget; we also internally allocated a certain number of mice per group and compared this to other studies with similar orientation to ensure adequate power of the study is achieved within the limitations of the cost.
Prednisolone was selected as a positive control group.
The nine groups were as follows Table
Experimental TNBS colitis in 62 male Wistar rats treated with CSF, GM-CSF and prednizolone, divided into 9 groups according to our experimental protocol.
Group | Number of animals with TNBS colitis | Day of euthanasia | Treatment with CSF (started) | Treatment with rHu GSF (started) | Treatment with prednisolone (started) |
---|---|---|---|---|---|
1 | 10 (control group, no treatment) | 15th | |||
2 | 10 (control group, no treatment) | 30th | |||
3 | 6 | 19th | Immediate treatment | ||
4 | 6 | 19th | 7 days after induction of colitis | ||
5 | 6 | 19th | 14 days after induction of colitis | ||
6 | 6 | 19th | Immediate treatment | ||
7 | 6 | 19th | 7 days after induction of colitis | ||
8 | 6 | 14 days after induction of colitis | |||
9 | 6 | 15th | Immediate treatment |
Growth factors were administered every 2 days at a dose of 10 mcg/kg. The dose of prednisolone was 5.3
The resected tissue samples were fixed in 10% buffered formaldehyde, and each specimen was entirely embedded in 3–4 paraffin blocks according to the size. Histological sections from each block were cut in 4
The malondialdehyde
The descriptive data were presented as mean ± standard deviation. Statistical comparisons between groups were made by Pearson’s chi square test or Student’s
All animals survived during the period of experiment.
Pathology results divided in three categories, 0: normal mucosa, 1: colitis in remission, 2: active colitis, according to Geboes histological score.
Groups | Category 0 (normal mucosa) | Category 1 (colitis in remission) | Category 2 (active colitis) | Pearson’s |
---|---|---|---|---|
1 (Colitis, 15 days, control group) | 20% (2/10) | 30% (3/10) | 50% (5/10) | |
2 (Colitis, 30 days, control group) | 50% (5/10) | 30% (3/10) | 20% (2/10) | |
3 (Granulocyte) | 67% (4/6) | 33% (2/6) | 0% (0/6) |
|
4 (Granulocyte) | 83% (5/6) | 17% (1/6) | 0% (0/6) |
|
5 (Granulocyte) | 83% (5/6) | 17% (1/6) | 0% (0/6) |
|
6 (Mielogen) | 50% (3/6) | 17% (1/6) | 33% (2/6) |
|
7 (Mielogen) | 0% (0/6) | 50% (3/6) | 50% (3/6) | NS |
8 (Mielogen) | 17% (1/6) | 0% (0/6) | 83% (5/6) | NS |
9 (Prednizolone) | 33% (2/6) | 50% (3/6) | 17% (1/6) |
|
Differences between CSF and prednizolone were statistically significant in favor of CSF (
Some representative histological pictures of the various animal groups are shown in Figures
Histological features in Groups 1, 2, 3 depicting lesions of ulcers with surrounded granulation tissue, normal mucosa or healing sites (colitis in remission). (a) extensive chronic ulcer (H/E ×25). (b) Ulcer (arrow) and regenerating adjacent epithelium (arrows) (H/E ×100). (c) Base of ulcer covered by granulation tissue (arrows) (H/E ×200). (d) Normal mucosa (arrow) and acute ulcer (arrows). (e) Healing site of previous ulcer (H/E ×200). Reepitheliastion (arrow) loss of crypts and fibrosis (arrows). (f) Healed site and regenerating crypts (arrow heads) reepithelialization (arrows) (H/E ×100).
Histological features in Groups 4, 5, 6, 7, 8, 9 depicting healing sites (colitis in remission), cryptic abscess (acute colitis), or chronic ulcers (chronic colitis).
The levels of tissue MDA in the treated and untreated animals are shown in Table
Mean value of tissue malondialdehyde in treated and untreated groups of animals and 95% confidence interval (mean ± SD, CI) (Comparisons: treated animals versus group 1).
Group | Tissue malondialdehyde ( |
|
---|---|---|
1 | 2.74 |
|
2 | 2.03 |
|
3 | 1.12 |
0.017 |
4 | 1.62 |
0.011 |
5 | 1.68 |
0.030 |
6 | 1.71 |
0.044 |
7 | 2.11 |
0.272 |
8 | 1.98 |
0.316 |
9 | 1.72 |
0.023 |
A significant reduction of tissue MDA in treated animals in groups 7 and 8 versus group 1 (control group), was noticed (
The results of the present study showed that the two growth factors namely Granulocyte-Colony Stimulating Factor (G-CSF,
Today, treatment of CD with granulocyte colony-stimulating factors should be considered as experimental treatments based on both experimental and clinical studies[
Experimental studies dated back to 1996 suggested that prolonged high-dose therapy with G-CSF may have anti-inflammatory effects in colitis [
Human studies have also described beneficial effect of various growth factors including growth hormone, keratinocyte growth factor (KGF), epidermal growth factor (EGF), teduglutide, and GM-CSF/G-CSF in patients with CD. Dejaco et al. found that G-CSF might be efficacious in severe endoscopic postoperative recurrence of CD. Another study showed that patients who were treated with 300
Korzenik et al. conducted a 12-week open-label trial with filgrastim in 20 patients with moderate to severe CD. Primary end-point was a decrease in the CDAI of >70 points and remission was considered to be a CDAI <150 points. All patients received filgrastim daily for 12 weeks at an initial dose of 300
GM-CSF has also been tested in patients with active CD. In an open-label dose-escalation trial (4–8 microg/kg/d) 15 patients with moderate to severe CD exhibited a significant decrease in the mean CDAI score by 190 points after 8 weeks of treatment. Overall, 12 patients had a decrease in CDAI of more than 100 points, and 8 achieved clinical remission [
Subsequently, Korzenik et al. investigated the role of sargramostim in 124 patients with CD. Patients were randomly assigned to receive sargramostim (6
In another clinical trial, patients with corticosteroid-dependent Crohn’s Disease were randomised to receive either sargramostim 6 microg/kg sc once daily or placebo for up to 22 weeks. Significantly more sargramostim-treated patients than placebo achieved corticosteroid-free remission as well as corticosteroid-free response[
Magno et al. evaluated the safety and efficacy of sargramostin in patients with fistulizing CD who had not responded to conventional therapy or had developed adverse reaction to infliximab. They found that rHu GM-CSF in a dose of 6 microg/kg/d sc for 8 weeks failed to improve the patients. However, the small number of patients and the severity of their disease do not allow drawing firm conclusions about the drug effectiveness [
However, a Cochrane meta-analysis of the three available studies with 537 patients showed that sargramostim does not appear to be more effective than placebo for induction of clinical remission or clinical improvement in patients with active CD [
Concerning other growth factors, it was suggested that keratinocyte-like growth factor-2 and epidermal growth factor enemas in combination with oral mesalamine, human growth hormone, and sargramostim may have significant synergistic effect in IBD patients [
As far as the possible mode of action of these agents is concerned, it has been proposed that growth factors could achieve both healing of the mucosa and restoration of the integrity of epithelium as well [
It is well established that the presence of neutrophils among epithelial cells is one of the major features of the inflammation in IBD patients, and has been used to indicate disease activity. The survival of neutrophils outside the blood vessels is limited and their longevity is influenced by GM-CSF, which probably reduces neutrophil apoptosis. Moreover, Griseri et al. emphasized the role of eosinophils as important effectors of IL-23 GM-CSF axis in colitis, the GM-CSF being a strong activator of eosinophilic functions [
Overall, our current study aims to shed light on the mechanisms of reduction of oxidative stress in all groups of treated animals, irrespectively of treatment (CSF, GSF, prednizolone). This was achieved by demonstrating reduced levels of tissue malondialdehyde. Although unknown, it could be related to avoidance of oxidant-induced mitochondrial injury [
This study has also some limitations. First, the number of the mice in each group was relatively small. Furthermore, the cost of purchasing mice is high and most previous studies have also used relatively small and similar groups.
Finally, another limitation of our study, might be the time of observation which is not related to the clinical outcome.
In conclusion, the administration of G-CSF and GM-CSF can significantly improve the histological score in experimentally induced colitis in rats. Both factors must be promptly administered in order to achieve maximum therapeutic response. Despite their high economic cost we suggest that these factors could further be tested in patients with acute exacerbation of IBD.
In future clinical trials must be administered as early as possible after the establishment of the acute flare-up of CD.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
JKT, AEP: conception and design of the study. MK, MS, DC: acquisition of data, drafting the article. CB, AN: histological study, analysis of histological scores. ET, DC, TP: drafting the article, revising it critically for important intellectual content. JKT, AEP: final approval of the version to be submitted.
The authors wish to thank Dr Aristofanis Gikas who performed the statistical analysis of the data and Eleftheria Karampela, Kalliopi Tsarea for their assistance in the laboratory during the experiments. Preliminary results of this study had been presented as a Poster in IBD 2007 Congress, “Achievements in Research and Clinical Practice” that was held in Istanbul in May 2007. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.