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. 2002 Jun;128(3):504–510. doi: 10.1046/j.1365-2249.2002.01838.x

Macrophage migration inhibitory factor activates antigen-presenting dendritic cells and induces inflammatory cytokines in ulcerative colitis

H MURAKAMI 1, S M F AKBAR 1, H MATSUI 1, N HORIIKE 1, M ONJI 1
PMCID: PMC1906246  PMID: 12109441

Abstract

The level of macrophage migration inhibitory factor (MIF) and the functions of dendritic cells (DC) are up-regulated in the peripheral blood, and the numbers of MIF-expressing cells and mature DC are increased at the colonic mucosa from patients with ulcerative colitis (UC). However, a functional relationship between MIF and DC, and the role of MIF in the pathogenesis of UC, are not clear. In this study, we showed that a pure population of peripheral blood DC is a new and still unknown source of MIF. DC from UC patients produced significantly higher levels of MIF (17·5 ± 9·8 ng/ml, n = 10) compared with patients with Crohn’s disease (CD) (4·6 ± 2·5 ng/ml, n = 5, P < 0·01) and control subjects (5·0 ± 2·6 ng/ml, n = 10, P < 0·01). A double immunofluorescence study revealed the expression of MIF by CD83-positive mature DC at the colonic mucosa from UC patients. Blood DC treated with high amounts of MIF (500 ng/ml) showed a significantly higher stimulatory capacity (43287 ± 5998 CPM, n = 5) in an allogenic mixed leucocyte reaction compared with untreated DC (27528 ± 8823 CPM, n = 5, P < 0·05). Study of intracellular cytokine expression showed that MIF induced significant levels of interleukin (IL)-1β and IL-8 in monocytes and DC from UC and CD patients. These results showing the capacity of MIF to induce increased functional capacity of DC, and to produce IL-1β and IL-8 from monocytes and DC, indicate a role of MIF in the induction and/or perpetuation of the inflammatory environment in UC.

Keywords: ulcerative colitis, MIF, dendritic cells, cytokines

INTRODUCTION

Ulcerative colitis (UC) is an inflammatory disease of the colonic mucosa and seems to result from a complex series of interactions among susceptibility genes, the environment and the immune system [1, 2]. Genetically-susceptible individuals, or persons with an alteration of the mucosal immune system towards luminal antigens, are prone to develop UC. However, the final assault for initiation and/or perpetuation of this disease mainly comes from inflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β, IL-6 and IL-8 [3–5]. These proinflammatory cytokines are produced mainly by activated lymphocytes and macrophages. However, little is known about the cellular events that lead to activation and recruitment of these cells in UC. As the antigen-presenting dendritic cells (DC) activate the lymphocytes and ensure the survival and functioning of activated lymphocytes [6, 7], and macrophage migration inhibitory factor (MIF) inhibits the random migration of macrophages in vitro and promote macrophage accumulation in vivo[8], we conducted two separate experiments to study the role of DC and MIF, if any, in the pathogenesis of UC. The results showed that the functional capacity of peripheral blood DC and the numbers of mature and activated DC at the colonic mucosa were significantly increased in patients with UC compared with normal healthy controls and patients with ischemic and inflammatory colitis [9]. We further showed that the levels of MIF in the sera were significantly increased in patients with UC, and MIF-expressing cells were detected at the colonic mucosa in UC [10]. Although these studies indicate that activated DC and increased levels of MIF may be related to the pathogenesis of UC, a functional relationship between DC and MIF, and the role of MIF in the induction of inflammatory cytokines in UC, has not been studied.

In the present experiments, we first studied the production of MIF by pure population of peripheral blood DC. We also checked the expression of MIF by DC at the colonic mucosa from UC patients and control subjects by double immunofluorescence methodology. Next, we evaluated the impact of MIF on the function of DC. Finally, the capacity of MIF to induce proinflammatory cytokines in T lymphocytes, monocytes and DC from normal subjects, UC and CD patients was assessed.

MATERIALS AND METHODS

Patients

Twenty-six patients with UC and 14 patients with Crohn’s disease (CD) attending the University Hospital of Ehime University between October 2000 and January 2002 were enrolled in this study. Twenty-five healthy volunteers served as controls. The diagnosis of UC and CD was based on the appropriate international criteria [1]. Ten patients with UC were maintained on prednisolone (5·0–40·0 mg/day), whereas 16 patients were not taking any immunosuppressive drug, including prednisolone. Five patients with CD were taking from 5–15 mg prednisolone daily and the others were not on any medication. The healthy control subjects were from volunteers and were not taking any medication during collection of blood. Informed consent was obtained from all patients and from the healthy volunteers, and this study was approved by the Human Research Committee of Ehime University.

Materials

RPMI 1640 (Iwaki, Chiba, Japan) plus 10% heat-inactivated fetal calf serum (FCS, Filtron Pty. Ltd, Brooklyn, Australia) containing streptomycin and penicillin was used as culture medium. Recombinant MIF was purchased from Genzyme/Techne (Cambridge, MA, USA). Brefeldin A (Sigma, St Louis, MO, USA) was used to block cytokine secretion from cytokine-producing cells. FACS lysing solution and FACS permeabilizing solution (Becton Dickinson Biosciences, San Jose, CA, USA) were used in intracellular cytokine assay. Peridin chlorophyll protein (PerCP)-conjugated monoclonal antibody (MoAb) to human CD3 (clone SK-7), CD14 (Clone MfP9), HLA DR (clone L243) and FITC-conjugated lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) were used to identify T cells, monocytes and DC during intracellular cytokine production (Becton Dickinson Biosciences). Phycoerythrin (PE)-conjugated antibody to human TNF-α (clone MAb11, BD Pharmingen, San Diego, CA, USA), IL-8 (clone AS11), IL-1β (clone AS10) and IL-6 (clone AS12) (all from Becton Dickinson Biosciences) were used to study the expression of these cytokines in different cells.

Isolation of peripheral blood mononuclear cells (PBMC), T cells, monocytes and DC

PBMC were collected from heparinized fresh blood by centrifuging on Ficoll-Conray (density 1·077) solution. The cells at the interface were washed and suspended in culture medium. Viability was checked by trypan blue exclusion (0·1% trypan blue).

T cells.T cells were isolated from PBMC using an affinity column (CollectTM, Biotex Laboratories INC, Edmonton, Canada) in which B cells were depleted from PBMC during their passage through the affinity column containing polyclonal goat anti-human IgG (H + L). Flow cytometric analysis revealed that the purity of T-cell populations was > 95%.

Monocytes.Monocytes were enriched from PBMC using a monocyte isolation kit (Miltenyl Biotech Gmbh, Bergisch Gladbach, Germany), in which CD3-, CD7-, CD19-, CD45RA-, CD56- and IgE-positive cells were depleted using magnetic cell sorting (MACS). The resultant cells were monocytes as confirmed by FACS analysis with PE-conjugated anti-human CD14 (Pharmingen) (purity >98%).

DC.Circulating DC in the peripheral blood were isolated from PBMC by two-step immunomagnetic cell sorting using a commercial kit, according exactly to the manufacturer’s instructions. Briefly, monocytes, T cells, B cells and natural killer cells were depleted from PBMC using MoAbs against CD3 (clone BW264/56), CD11b (clone M1/70.15.11.5) and CD16 (clone VEP-13) (Miltenyl Biotech Gmbh). From the depleted cell fraction, DC were then enriched using a MoAb against CD4 (clone M-T321, Miltenyl Biotech Gmbh). FACS analysis revealed that the contaminating T cells, B cells and NK cells were <1·0% in the DC population.

Production of MIF by T cells, monocytes and DC

Purified populations of T cells, monocytes and DC (1 × 106) were cultured in 1·0 ml culture medium at 37°C in a humidified atmosphere containing 5% CO2 in air. The clear supernatant fluids were collected, centrifuged and preserved at – 80°C. MIF in the culture supernatant fluid was estimated by a double sandwich enzyme-linked immnunosorbent assay (ELISA) method, using a commercial kit (IDLISA Human MIF immunoassay kit, Sapporo Immunodiagnostic Laboratory, Sapporo, Japan) according exactly to the manufacturer’s instructions, and has been described previously in detail [10]. In short, the culture supernatant fluids were allowed to react with mouse anti-human MIF monoclonal antibody on microtitre plates and then with horseradish peroxidase-labelled mouse anti-human MIF monoclonal antibody. Tetramethyl benzidine was used as chromogen and the optical density (OD) value at 450 nm was measured using an ELISA reader (SJeia Auto Reader, Model ER-8000, Sanko Junyaku Co. Ltd, Tokyo, Japan). A standard curve was made by plotting the OD values against the amount of MIF (ng/ml) in the standard sera. The detection level of MIF by this kit is ≥1·6 ng/ml.

Cell cultures

Activation of DC by MIF. For generation of MIF-activated DC, highly purified peripheral blood DC (1 × 106 cells/ml) were cultured with graded doses (1, 10, 100, 500 ng) of recombinant MIF for 24 h at 37°C in a humidified 5% CO2-containing atmosphere. DC were then washed twice with phosphate-buffered saline (PBS) and allowed to recover from the effect of MIF for some time. DC cultured in medium containing RPMI 1640 plus 10% FCS for 24 h were used as control DC.

Allogenic mixed leucocyte reaction (MLR). We have previously described the culture conditions for allogenic MLR in details [11]. T cells (2 × 105) from one allogenic normal control (MH, age 32 years, male) were cultured with γ-irradiated (40 Gy, Hiltex Co., Ltd, HW-150, Osaka, Japan) DC (1 × 104) for 5 days at 37°C in a humidified incubator containing 5% CO2 in air. The levels of incorporation of 3H]-thymidine during the last 16 h of the total 120 h culture were determined in a liquid scintillation counter (Beckman LS 6500, Beckman Instruments, Inc., Fullerton, CA, USA) as counts per minute (CPM).

Flow cytometric analysis of intracytoplasmic cytokine production

The expression of intracellular cytokines by T cells, monocytes and DC in peripheral blood due to stimulation with MIF was measured by flow cytometry according to previously described methodology [12]. PBMC (1 × 106/ml) were either stimulated with 100 ng MIF or placed unstimulated in the presence of 10 mg brefeldin A for 2–6 h at 37°C in a 5% CO2 humidified atmosphere. The samples were then aliquoted into different tubes and stained with 20 μl PerCP-conjugated MoAb to CD3 (for T cells), CD14 (for monocytes), HLA DR and lineage cocktail (containing CD3, CD14, CD16, CD19, CD20 and CD56) (for DC) for 15 min in the dark at room temperature. The cells were then incubated with FACS lysing solution (diluted 1:10) and subsequently, with FACS permeabilizing solution (1:10) at room temperature. Then, 20 μl PE-conjugated MoAb to TNF-α, IL-8, IL-1β and IL-6 were added to the respective tubes and incubated for 30 min in the dark at room temperature. Mouse IgG1 PE-conjugated MoAb was used as isotype-matched control antibody. The expression of various intracellular cytokines in T cells, monocytes and DC were analysed using FACS Calibur (Beckton Dickinson).

Immunofluorescence

We have previously described in detail the methodology for detection of CD83-positive mature DC [9] and MIF-expressing cells [10] at the colonic mucosa by immunohistochemistry from patients with UC and normal controls. Here, we used immunofluorescene methodology to detect MIF expression by CD83-positive DC. Mouse anti-human CD83 monoclonal antibody (clone HB15a, Immunotech, Marseille, France) and goat anti-human MIF (Genzyme/Techne, San Carlos, CA, USA) were used as primary antibodies. MIF-positive cells were detected by greenish staining of FITC, whereas the CD83-positive DC were localized by reddish staining of Cyanin (CY)-3. Cells co-expressing MIF and CD83 were stained yellowish.

Statistical analysis

The levels of MIF were shown as mean ± standard deviation (mean ± s.d.). Means were compared with the unpaired t-test. In cases of differences (as assessed by an F-test), t-tests were adjusted for unequal variances (Mann–Whitney’s U-test). P < 0·05 was considered to be statistically significant. Statistical calculations were performed using the Stat View (version 4·5) statistical programme in a Macintosh computer (Power Mac G4).

RESULTS

Peripheral blood DC as a new source of MIF

We studied the spontaneous production of MIF by peripheral blood DC, as well as T cells and monocytes, in 24 h cultures. As shown in Fig. 1, the mean levels of MIF produced by DC were significantly higher in UC patients (17·5 ± 9·8 ng/ml, n = 10) compared with control subjects (5·0 ± 2·6 ng/ml, n = 10, P < 0·01). However, the levels of MIF produced by CD patients were almost the same as those produced by normal controls (4·6 ± 2·5 ng/ml, n = 5). Eight of the 10 patients with UC produced >10·0 ng/ml MIF in culture, and none of these patients were taking prednisolone. One of the two patients with UC producing <10·0 ng/ml MIF was maintained on prednisolone (black square, Fig. 1). However, the levels of MIF produced by monocytes and T cells were similar amongst patients with UC and CD, and the normal control.

Fig.1.

Fig.1

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 Increased levels of MIF produced by DC from patients with UC. Highly purified population of DC, monocytes and T lymphocytes from control subjects (○), patients with UC (•) and CDInline graphicwere cultured for 24 h. A patient with UC maintained on prednisolone is shown by a black square (▪). Bar and lines indicate the mean ± s.d. in each group. *P < 0·01 compared with DC from control subjects and patients with CD.

To study the effect of prednisolone on MIF production by DC, we cultured DC with or without prednisolone sodium succinate (Shionogi Co, Osaka, Japan). DC cultured with a high dose of prednisolone (1 × 10−4 M) produced significantly lower levels of MIF compared with untreated DC (2·5 ± 0·8 versus 4·8 ± 1·6 ng/ml, n = 5, P < 0·05).

Expression of MIF by mature DC at the colonic mucosa in UC

The expression of MIF by CD83-positive mature DC at the colonic mucosa from a representative patient with UC is shown in Fig. 2. Cells expressing only MIF (greenish, shown by star) or only CD83 (reddish, shown by triangle) were seen at the colonic mucosa from patients with UC, along with some cells co-expressing CD83 and MIF (yellowish, shown by arrow head). Although cells expressing both CD83 and MIF were detected at the colonic mucosa from patients with UC (Fig. 2a and b), these cells were not found at the colonic mucosa from a normal control (Fig. 2c) and five patients with CD (data not shown).

Fig.2.

Fig.2

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 Expression of MIF by CD83-positive DC at the colonic mucosa from a patient with UC (a) and (b), but not at the colonic mucosa from a normal control (c). MIF-expressing cells are shown by a green signal of FITC (star) and CD83 was detected by reddish staining of Cy-3 (triangle). Cells expressing both MIF and CD83 are shown by yellowish signals (arrow head).

Activation of peripheral blood DC by MIF

As shown in Fig. 3, the levels of CPM in allogenic MLR containing DC treated with 500 ng/ml MIF were significantly higher (43 287 ± 5998 CPM, n = 5) compared with the levels of CPM in allogenic MLR containing untreated DC (27 528 ± 8823 CPM, n = 5, P = 0·0433, Mann–Whitney’s U-test) or DC treated with a low dose of MIF (1 ng/ml) (24 406 ± 7931 CPM, n = 5, P = 0·021, Mann Whitney’s U-test).

Fig.3.

Fig.3

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 Increased stimulatory capacity of DC treated with a high dose of MIF in allogenic MLR. Purified DC were cultured either in media without any MIF, or with different doses of MIF for 24 h. These DC were then challenged to stimulate allogenic T cells from a normal control volunteer. Data are shown as mean ± s.d. from five separate experiments. P < 0·05 compared with untreated DC and DC treated with 1·0 ng/ml MIF.

Induction of proinflammatory cytokines from monocytes and DC by MIF

A representative dot plot of intracellular expression of TNF-α, IL-1β, IL-6 and IL-8 of untreated and MIF-treated T cells, monocytes and DC are shown in Fig. 4. Very few T cells showed intracellular expression of any of these cytokines due to stimulation with MIF (Fig. 4). Similarly, a 2 h stimulation with MIF induced intracellular cytokines in very few DC, but a 6 h stimulation with MIF induced these cytokines in some DC (Fig. 4). Intracellular expression of these cytokines was detected in monocytes due to 2h and 6h stimulation with MIF.

Fig.4.

Fig.4

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 Intracellular cytokine production by T cells, monocytes and DC due to stimulation with MIF. Representative dot plots of TNF-α, IL-1β, IL-6 and IL-8 production by untreated and MIF-treated T cells, monocytes and DC in the presence of brefeldin A are shown.

As shown in Fig. 5, the numbers of monocytes expressing intracellular IL-8 due to 2h stimulation with MIF were significantly higher in patients with UC than in control subjects (P < 0·05). IL-8-expressing monocytes, in particular, were increased in UC patients who had not received prednisolone (open circles) therapy compared with patients treated with prednisolone (closed circles).

Fig.5.

Fig.5

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 Intracellular production of IL-1β and IL-8 by monocytes due to stimulation with 100 ng/ml of MIF for 2 h. (□) Control subject; (○) UC; (▵) CD. Black symbols indicate the patients receiving prednisolone therapy and the open symbols represent patients not receiving prednisolone therapy. Mean and s.d. are also shown as lines.

The mean numbers of DC expressing intracellular cytokines due to 6h stimulation with MIF are shown in Table 1. The numbers of DC expressing intracellular IL-1β were significantly higher in patients with UC than in control subjects (P < 0·05). IL-8-expressing DC were increased in UC and CD patients compared with control subjects (P < 0·05).

Table 1.

 Intracellular cytokine expression by DC

n TNF-α IL-1β IL-6 IL-8
Control 10 3·7 ± 1·3% 19·6 ± 6·9% 1·6 ± 0·8% 15·8 ± 3·9%
UC 10 6·0 ± 2·7% 27·3 ± 7·9%* 3·1 ± 2·3% 21·5 ± 6·5%*
CD 10 8·5 ± 4·2% 23·9 ± 5·5% 6·1 ± 5·0% 27·6 ± 10·0%*
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Intracellular expression of different cytokines in DC due to stimulation with 100 ng MIF for 6 h in the presence of brefeldin A. The data are shown as percentage of cells expressing intracellular cytokines (mean ± s.d.).

*

P < 0·05 compared with DC from control subjects.

DISCUSSION

Genetically-susceptible individuals or persons with an alteration of the mucosal immune system towards luminal antigens are prone to develop UC. However, inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IL-8 are thought to provide the final assault for initiation and/or perpetuation of mucosal damage in UC [1–5, 8 13). We have already shown that MIF, which inhibits the random migration of macrophages, is hyper-expressed, and antigen-presenting DC, which induces maturation of T cells and macrophages, are highly activated, both in the peripheral blood and at the colonic mucosa in UC patients, but not in patients with ischemic colitis, infective colitis and control subjects [9, 10].

In this study, we were able to show that, in addition to T cells [14], macrophages [15] and eosinophils [8], human peripheral blood DC are a new and still unknown source of MIF (Fig. 1). It may appear that MIF joins a list of several inflammatory cytokines produced by DC, such as IFN-α, IFN-γ, IL-1β and IL-8 [6, 7]. However, there is a fundamental difference between these findings and the study presented here. Prior to the development of the MACS technique, DC were enriched by culturing monocytes with IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) (monocyte-derived DC) for 6–8 days [6, 7, 9, 11]. Thus, it was unclear whether the cytokines produced by DC were really DC-derived cytokines or whether they were products of monocytes. Also, the sustained presence of IL-4 and GM-CSF in these cultures could alter the cytokine profiles. DC used in the present experiment were almost completely free from contamination by T cells, B cells, monocytes and NK cells, and we cultured these DC without any stimulant to evaluate MIF production. The amounts of MIF produced by circulating peripheral blood DC from UC patients were significantly higher than the amounts of MIF produced by DC from control subjects and patients with CD (Fig. 1). We also found expression of MIF by some mature DC at the colonic mucosa from UC patients (Fig. 2c,d) using an immunofluorescence technique. MIF caused activation of DC in vitro (Fig. 3) and induction of IL-1β and IL-8 in monocytes and DC from UC patients. These findings indicate a role for MIF during the induction and/or perpetuation of colonic mucosal damage in UC [1–5, 8, 13]. Furthermore, MIF induced IL-8 in DC from CD patients. MIF may be involved in the inflammatory environment in CD through the production of IL-8 by DC.

Data from this study indicate that steroid could have an inhibitory effect on MIF production in UC (Fig. 1, Fig. 5). The suppressive effect of steroid on MIF production from lymphocytes has been described by Bucala [16]. Here, we showed that steroid down-regulated the MIF-producing capacity of DC. Steroid may act directly on DC to down-regulate MIF production. In addition, steroid may induce some inhibitory mediators which may down-regulate the production and activity of MIF. A study is in progress in this laboratory on the role of IL-10 and TGF-β in MIF production from DC in the presence of steroid.

The present study provides some insight into the inflammatory mucosal milieu in UC. It is now widely accepted that both inflammatory cytokines [1–5, 8 13] and the inflammatory mucosal milieu [17] play a role in the initiation and/or perpetuation of UC. Indeed, several inflammatory cytokines have been shown to be up-regulated in UC patients [1–5, 8 13]. Regarding the inflammatory milieu, Bell et al. made a very interesting observation in that many apoptotic T cells were detected at the colonic mucosa from normal subjects, but not in UC [18]. High levels of MIF in UC may immobilize macrophages and facilitate their localization at the colonic mucosa, leading to production of several inflammatory cytokines. MIF may also cause activation and maturation of DC. These activated and mature DC, along with the inflammatory cytokines, may play a role in the prolonged survival of activated T cells and maintenance of the inflammatory mucosal milieu in UC. The next important step would be to clarify whether down-regulation or neutralization of MIF would reverse these inflammatory mucosal milieu in UC patients. It has already been shown that neutralization of MIF by anti-MIF has a potent anti-inflammatory effect in many pathological conditions [19, 20], including severe inflammatory conditions such as fulminant hepatitis [21]. A study is in progress in our laboratory on the effect of neutralization of MIF in UC.

In summary, we have shown that DC are a new and unknown source of MIF, and both the peripheral blood DC and colonic DC from patients with UC produce MIF. MIF also induces activation of DC and inflammatory cytokines such as IL-1β and IL-8 from monocytes and DC. Taken together, these studies show a role of MIF in the inflammatory mucosal milieu in UC, and inspire optimism for a therapeutic role of neutralization of MIF in UC patients.

Acknowledgments

The authors are grateful to Kenji Tanimoto and Ayumi Sone, Third Department of Internal Medicine, Ehime University School of Medicine, Japan for their assistance in immunohistochemistry.

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