Immune system alterations in patients with inflammatory bowel disease during remission.

OBJECTIVE
Perturbed immune homeostasis elicited by misbalanced production of proinflammatory and anti-inflammatory cytokines is characteristic of inflammatory bowel disease. The aim of this study was to evaluate cytokine profile in patients with different forms of inflammatory bowel disease - ulcerative colitis and Crohn's disease - during clinical remission phase.


MATERIAL AND METHODS
Production of proinflammatory Th1 cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)) and anti-inflammatory Th2 cytokines (interleukin-10 (IL-10) and interleukin-13 (IL-13)) was analyzed in peripheral blood mononuclear cells of patients with inflammatory bowel disease (9 with ulcerative colitis and 9 with Crohn's disease) and control subjects (n=11) by enzyme-linked immunosorbent assay (two-site ELISA).


RESULTS
The results of the study revealed that the level of TNF-alpha after stimulation with phytohemagglutinin in patients with Crohn's disease was significantly higher in comparison to both patients with ulcerative colitis and controls (P<0.001 and P<0.01, respectively). The secretion of IFN-gamma both in patients with Crohn's disease and ulcerative colitis was lower than that in controls (P=0.05 and P<0.01, respectively), but it normalized after stimulation with phytohemagglutinin. The levels of IL-10 and IL-13 were significantly (P<0.01) higher in patients with Crohn's disease than in patients with ulcerative colitis and control group before and after stimulation with phytohemagglutinin.


CONCLUSIONS
The results of our study provide evidence that in patients with inflammatory bowel disease, the imbalance between production of proinflammatory Th1 and anti-inflammatory Th2 cytokines persists even during remission of the disease, and disturbances of immune homeostasis are significantly more expressed in patients with Crohn's disease than in patients with ulcerative colitis.


Introduction
Ulcerative colitis (UC, OMIM 191390) and Crohn's disease (CD, OMIM 266600), collectively referred to the inflammatory bowel disease (IBD), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles (1)(2)(3).The excessive production of either Th1 or Th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes, and loss of immune homeostasis may be implicated in the genesis of IBD (4)(5)(6).Although the etiology of IBD is still unknown, the animal model and human case-control studies have revealed that in the intestinal tissue and peripheral blood during active CD phase, the levels of tumor necrosis factor-alpha (TNF-a), interferongamma (IFN-g), interleukin (IL)-12 and IL-1 are increased, whereas in UC, the secretion of IL-5, IL-4, IL-10, and IL-13 is upregulated, elucidating that immune mechanisms playing a major role in the initiation and perpetuation of both diseases are distinct.Thus, CD is characterized by the typical Th1 cell response and UC by an atypical Th2 cell response (7)(8)(9)(10)(11), whereas cytokine profile in IBD patients during the remission phase has not been investigated thoroughly yet.
Therefore, the aim of our study was to explore pro-and anti-inflammatory cytokine profile in patients with IBD during the remission of the disease.We used cells of the immune system that are easily accessible in the peripheral blood and can be studied ex vivo and investigated spontaneous and stimulated production of TNF-a, IFN-g, IL-10, and IL-13 in patients with two different IBD forms and in healthy control subjects.

Patients
Blood samples were collected from 18 IBD patients in remission phase (9 CD patients (men/women, 4/5; mean age, 37.08 years; range, 24-65 years), 9 UC (men/women, 4/5; mean age, 37.00 years; range, 21-55 years)) and 11 unrelated healthy, age-and sexmatched controls.The recruitment of study individuals was performed at the Department of Gastroenterology, Kaunas University of Medicine Hospital.The diagnosis of either CD or UC was based on standard clinical, endoscopic, radiological, and histological criteria (12).For the assessment of disease activity, colitis activity index (CAI) according to Gomes et al. (13) and CD activity index (CDAI) (14) were used.Only patients with inactive disease (CAI<4 and CDAI<150) and using 5-aminosalicylate (5-ASA) preparations as maintenance therapy were included into the study.The patients receiving immunosuppressive therapy or treated surgically were excluded from the study.Written informed consent was obtained from all study participants.The study had been approved by the Kaunas Regional Biomedical Research Ethics Committee (Protocol No. BE-2-49).

Cell collection
Peripheral blood mononuclear cells (PBMNC) were separated from EDTA-treated peripheral blood (10 mL) by a standard procedure using Ficoll density gradient centrifugation.After three washes in serumfree RPMI-1640 medium (Biochrom, Germany), the mononuclear cells were resuspended in complete growth medium (RPMI supplemented with 2 mM L-glutamine, antibiotics (100 U/mL penicillin and 100 mL streptomycin) and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany)) and processed cytokine assay as described below.

Cytokine assay
For cytokine assay, cells were adjusted to a concentration of 1×10 6 cells per mL and cultured in complete growth medium in 24-well plates at a 5% CO 2 and 37°C humidified atmosphere (Incubator IR AUTO-FLOW Water Jacketed, USA).Cell culturing was performed without and with addition of phytohemagglutinin (PHA; 5 mg/mL) (Biochrom, Germany).After 24 hours, supernatants were collected and stored at -20°C until assay.Concentrations of cytokines TNF-a, IFN-g, IL-10, and IL-13 in supernatants were determined with commercially available two-site ELISA kits (Amersham Biosciences, UK).The lowest limit of sensitivity of test systems for TNF-a was 4 pg/mL; for IFN-g, <2 pg/mL; for IL-10, <3 pg/mL, and for IL-13, <7 pg/mL.The optical densities at 450 nm and at a correction wavelength of 620 nm were measured on a microplate reader (DigiScan 400, Asys Hitech).

Statistical analysis
Statistical analyses were done using the SPSS statistical package (version 12.0; Chicago, IL).The data in text, table, and figures are presented as means±SE.The difference between two independent samples was compared using Mann-Whitney U test.An analysis of variance (ANOVA one-way) was used to compare data on immunological analysis between different study groups.A P value of <0.05 was accepted as statistically significant.

Proinflammatory cytokines
The spontaneous secretion of proinflammatory TNF-a cytokine was highest in CD and lowest in UC patient groups (Table, Fig. 1).The difference between two IBD groups was statistically significant (P=0.02).After stimulation with PHA, the production of TNF-a increased from 20-to 30-fold in all study groups and retained the same production patterns as in cells cultivated without PHA; however, the cytokine level in CD patients became significantly higher not only in comparison to UC patients, but also to controls.The concentrations of IFN-g (Table, Fig. 2) in both IBD patient groups were lower than in controls (P<0.01 for UC; P=0.05 for CD).However, the difference disappeared after the stimulation with PHA.

Anti-inflammatory cytokines
The level of the production of anti-inflammatory cytokine IL-10 was highest in CD patients (Table, Fig. 3), and the difference was significant in comparison to UC and control subjects (P<0.001 and P=0.02, respectively).The difference became even more prominent after the stimulation with PHA.In UC patients, IL-10 level before stimulation was lower than in controls (P<0.001);however, the difference disappeared after cultivation of cells with PHA.
In CD patients, the concentration of IL-13 before stimulation was significantly higher when comparing with UC patients and controls (P<0.001),and this pattern was retained after the stimulation of cells with PHA (Table, Fig. 4).In UC subjects, the level of spontaneous IL-13 production was similar to the levels observed in the control group; however, after the Immune system alterations in patients with inflammatory bowel disease during remission

Fig. 2. Level of IFN-g (pg/mL) in peripheral blood mononuclear cells cultivated without (white columns) and with phytohemagglutinin (PHA) (dark columns) in patients with Crohn's disease (CD) and ulcerative colitis (UC) and control group
Data are presented as mean±SE.

Fig. 3. Level of IL-10 (pg/mL) in peripheral blood mononuclear cells cultivated without (white columns) and with phytohemagglutinin (PHA) (dark columns) in patients with Crohn's disease (CD) and ulcerative colitis (UC) and control group
Data are presented as mean±SE.
stimulation, the cytokine concentration became significantly higher than in controls.

Discussion
It is generally accepted that the excessive production of either pro-and anti-inflammatory cytokines The increased production of Th1 cytokines has been established to be a feature of active CD but not of UC (7)(8)(9)(10)(11)15), while the number of studies analyz-ing the generation of the proinflammatory subset of cytokines (especially in PBMNC) during IBD remission is limited (16)(17)(18)(19).The results of our study revealed that the concentration of TNF-a in both CD and UC patients has not differed from controls; however, the stimulation with PHA significantly increased the production of TNF-a in CD patients.The secretion of IFN-g in both IBD groups was even lower than that in controls, and the difference disappeared after the stimulation.Normal (TNF-a) or even hyposecretion (IFN-g) of proinflammatory Th1 cytokines in CD patients with inactive disease is consistent with findings reported by other authors (16,17).However, our data revealed that the stimulation with PHA caused a significant increase in TNF-a production, typical for CD, even in case of inactive disease.Hyposecretion of IFN-g in IBD patients may be related to the refractoriness of T cells that have been continuously activated in vivo as proposed by Wilde et al. (20) or to the down-regulation of the proinflammatory cytokines as the result of therapy with 5-ASA preparations, which also have the suppressive effect on the immune system (21,22).
The existence of the Th2-biased pattern production in UC but not CD during active state of disease has been confirmed by some studies (7)(8)(9)(10)(11), whereas there are only scarce data on cytokine profile in case of noninflammatory or inactive disease (22,23).In our study, we found the increased production of IL-10, which is critical for temporizing the proinflammatory processes via strong down-regulation of Th1 cytokines (15,16) during remission phase in CD patients compared with UC patients and control group.These results oppose to recently published publication by Mitsuyama et al. (23), indicating the increased secretion of IL-10 during disease recovery in patients with UC, but not CD.Our study is the first report analyzing IL-13 levels in IBD patients during remission phase.We found the increased production of this cytokine in CD group as compared to UC and control groups.Anti-inflammatory cytokines have been reported to have inhibitory effects on activated macrophages producing different cytokines and effector functions on monocytes and T cells (24)(25)(26).CD during relapse phase has been reported to have a proinflammatory ("Th1-like") cytokine profile (7)(8)(9)(10)(11); therefore, the up-regulation of the anti-inflammatory cytokines seems to be due to response to proinflammatory cytokine activity.The data of our study suggest that IL-10 and IL-13 act as natural dampers of immune proliferation and inflammatory responses.However, it has already been ascertained that an excessive production of either proinflammatory or anti-inflammatory cytokines may also result in the inappropriate suppression of the immune response and may provoke a compensatory reaction (11).
Although the data in our study were obtained from isolated peripheral blood monocytes, relevance to the gut may be inferred since peripheral blood monocytes are precursors of tissue macrophages.Monocytes thus provide an opportunity to study the mononuclear phagocyte cell line before the monocytes migrate from the blood into the bowel mucosa where they became activated.These immunological responses seen in the peripheral blood are events that mirror systemic activation in the gut (27).

Conclusions
The results of our study provide evidence that in patients with inflammatory bowel disease, the disso-nance between production of proinflammatory Th1 and anti-inflammatory Th2 cytokines persists even during the remission of the disease, and disturbances of immune homeostasis are significantly more expressed in patients with Crohn's disease than in patients with ulcerative colitis.

Fig. 1 .
Fig. 1.Level of TNF-a (pg/mL) in peripheral blood mononuclear cells cultivated without (white columns) and with phytohemagglutinin (PHA) (dark columns) in patients with Crohn's disease (CD) and ulcerative colitis (UC) and control group Data are presented as mean±SE.Mean concentration (pg/mL) Mean concentration (pg/mL) spontaneous TNF-a production PHA-stimulated TNF-a production role in immune and inflammatory reactions and loss of immune homeostasis in patients with IBD(7)(8)(9)(10)(11).

Fig. 4 .
Fig. 4. Level of IL-13 (pg/mL) in peripheral blood mononuclear cells cultivated without (white columns) and with phytohemagglutinin (PHA) (dark columns) in patients with Crohn's disease (CD) and ulcerative colitis (UC) and control group Data are presented as mean±SE.