INTRODUCTION

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Macrophage migration inhibitory factor (MIF) was first described in 1966 as a cytokine "activity," derived from activated T lymphocytes, preventing random macrophage migration at the site of inflammation (1, 2). After the cloning of human MIF complementary DNA (cDNA) in 1989 (3), MIF "protein" was re-evaluated as an important pro-inflammatory cytokine (4, 5). In a series of recent studies using rodents, MIF was found to be a secretory product of anterior pituitary cells and activated macrophages that antagonizes the suppressive effects of glucocorticoids on cytokine production and potentiates effects of endotoxin in experimentally induced septic shock (6). Furthermore, administration of an anti-MIF antibody improves the survival rate in the case of lethal endotoxemia in mice (4, 5). Since then, a few laboratories, including ours, have reported that MIF can be detected in a variety of cells, tissues, and organs in humans as well as in rodents (7), and that MIF production is upregulated in gram-negative sepsis (11). More recently, Donnelly and colleagues (12) demonstrated that in patients with acute respiratory distress syndrome (ARDS), MIF is present in the affected lungs, and that alveolar macrophages are one cellular source of MIF. They also showed that MIF augments, and the anti-MIF antibody attenuates, pro- inflammatory cytokine secretions, such as tumor necrosis factor alpha (TNF-) and interleukin-8 (IL-8) from alveolar macrophages.

This background prompted us to examine the protective effect of an anti-MIF antibody on lipopolysaccharide (LPS)- induced acute lung injury in rats. We anticipated that pretreatment with the anti-MIF antibody could attenuate or abolish such lung injury. Additionally, we investigated the localization of MIF in the lungs of rats by immunohistochemical analysis, and the expression of MIF mRNA before and after the LPS-induced lung injury by northern blot analysis. Furthermore, in an attempt to gain insight into the mechanism by which the anti-MIF antibody attenuates LPS-induced neutrophil accumulation in the lungs, we measured macrophage inflammatory protein-2/cytokine-induced neutrophil chemoattractant (MIP-2/CINC-3), an important neutrophil chemokine, in bronchoalveolar lavage (BAL) fluid of the rats.

	METHODS

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Animals

Studies were performed on male Sprague-Dawley rats (n = 69) (224 ± 52 g [mean ± SD]). They were allowed free access to water and commercial chow. This research adhered to the Declaration of Helsinki and was approved by the Animal Experiment Ethics Committee of Hokkaido University School of Medicine.

Materials

The following materials were obtained from commercial sources: LPS from Escherichia coli 0111:B4 was purchased from Sigma Chemical Company (St. Louis, MO) and dissolved in pyrogen-free physiologic saline just before each experiment; biodyne transfer membranes from Pall Biosupport Division (Glen Clove, NY); Isogen RNA extraction kit from Nippon Gene Chemical Company (Tokyo, Japan); [-³²P] dCTP from Dupont-NEN (Boston, MA); complete Freunds' adjuvant (CFA) and incomplete Freunds' adjuvant (IFA) from Wako Pure Chemical Industries (Osaka, Japan); Vector ABC Kit from Vector Laboratory (Burlingame, CA); horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody from Pierce (Rockford, IL); specific rat MIP-2/CINC-3 ELISA kit from Immunobiological Laboratory (Fujisawa, Japan). All other chemicals were of analytical grade.

Preparation of Rabbit Polyclonal Antibody against Rat MIF

Polyclonal anti-rat MIF serum was generated by immunizing New Zealand White rabbits with purified recombinant rat MIF. Rat MIF was expressed in E. coli and purified to homogeneity as described in our previous publications (13, 14). In brief, the rabbits were inoculated intradermally with 100 µg of MIF emulsified in CFA at Weeks 1 and 2, and with 50 µg of MIF diluted in IFA at Week 4. The immunoglobulin G (IgG) fraction was prepared using Protein A-Sepharose according to the manufacturer's protocol.

Experimental Design

Experiments were designed to evaluate the effects of the anti-MIF antibody on LPS-induced acute lung injury by comparing an LPS group (nonimmunized rabbit IgG + LPS) and an anti-MIF Ab group (anti-MIF antibody + LPS) 4 and 24 h after LPS administration. LPS was administered intraperitoneally (7 mg/kg of 2 mg/ml LPS in 0.9% NaCl). As a negative control, physiologic saline, instead of LPS, was given to rats intraperitoneally. Two hours before administration of LPS, the rats were intraperitoneally injected with either the anti-MIF antibody (IgG fraction; 3.9 ~ 8.6 mg/kg) or the same dose of nonimmunized rabbit IgG. The rats were then returned to their cages and allowed free access to food and water. Four and 24 h later, blood samples were taken by puncturing the abdominal aorta under anesthesia with 1.5% inhalant halothaine. Then the lungs were excised by opening the chest, and free blood was removed by blotting the hilus onto paper towels. The left lung was fixed in buffered (pH 7.4) 10% formalin for at least 48 h and then embedded in paraffin for later histopathologic examination. The upper part of the right lung was used for the measurement of tissue myeloperoxidase (MPO) activity, and the lower part of the right lung was used for obtaining the lung tissue wet to dry (W/D) weight ratio.

Histologic examination. A 5-µm section was cut from the mid-portion of paraffin-embedded whole lung tissue and stained with hematoxylin and eosin. An observer who was blinded to the animals' group assignment assessed more than 50 alveoli at ×400 magnification and determined the average number of neutrophils per alveolus.

Measurement of the lung tissue MPO activity. The lung was homogenized and sonicated in 100 mM potassium phosphate-buffered solution (PBS), pH 6.0, with 0.5% hexadecyl-trimethylammonium bromide (HTAB) and 5 mM EDTA. After centrifugation (40,000 × g, 15 min), the supernatant fluids were reacted with H₂O₂ (0.0005%) in the presence of orthodianisidine (0.167 mg/ml). The change in optical density (OD) (at 460 nm) per minute was determined (15).

Measurement of the lung tissue W/D weight ratio. The lung was weighed soon after excision, and then dried in an oven at 60° C for 72 h. The dried lung tissue was weighed again, and the lung tissue W/D weight ratio was obtained.

Neutrophil counts and albumin concentration in BAL fluid. Another set of animals was used for evaluation of cell differentiation and the albumin concentration in BAL fluid. BAL was performed under anesthesia with intraperitoneally administered sodium pentobarbital (50 mg/kg) 4 and 24 h after the administration of LPS. Each time, 8 ml of 37° C sterile physiologic saline was instilled through a tracheal cannula at a hydrostatic pressure of 15 cm and withdrawn by gravity while using a vibrator. The procedure was repeated five times, and all BAL fluid was collected. The recovery rate was over 90%. Then we centrifuged the BAL fluid (700 × g for 5 min, 4° C) and counted the total number of cells using a standard hemocytometer method. We stained the cells by the Dif-Quik method and determined the cell differentiation.

Immunohistochemistry

The lungs of rats that had received either LPS or physiologic saline (n = 3 for each) 24 h earlier were used for the immunohistochemical study. The lungs were fixed overnight in formaldehyde, after which 5-µm thick parafilm sections were mounted on poly-L-lysine-coated slides. The BAL cells were collected as described above (n = 3 for each), and mounted on poly-L-lysine-coated glass slides. After centrifugation for mounting, the BAL cells were fixed in 95% ethanol at 4° C for 5 min. The tissue samples were immersed in 100% methanol containing hydrogen peroxide (0.3%) for 30 min to quench endogenous peroxidase reactivity. Subsequently, they were stained with the avidin-biotin-peroxidase complex procedure using a Vector ABC Kit according to the manufacturer's protocol. Nonspecific staining was blocked by incubation for 30 min with normal goat serum (10%). The sections were further incubated overnight at 4° C with the anti-rat MIF polyclonal antibody. After three washes with PBS, the samples were reacted with biotinated goat anti-rabbit IgG and avidin-biotin complex at room temperature for 30 min. The reaction was developed in 3,3'-diaminobenzidine tetrahydrochloride containing hydrogen peroxide (0.01%), and the tissue samples were mounted with alkylacrylates.

Northern Blot Analysis

Lung tissues were collected under anesthesia before and 24 h after LPS administration (n = 3 for each). Northern blot analysis was carried out as previously described (7). In brief, RNA from these lung tissues was extracted and separated into 20-µg units by electrophoresis on 1% agarose gel containing 0.6 M formaldehyde, and blotted on nylon membrane filters. The hybridization was carried out using a full-length rat MIF cDNA probe radiolabeled with [-³²P]dCTP at 42° C for 48 h. The MIF cDNA used in this study had been isolated from a rat liver cDNA library in this laboratory (7). After hybridization the filters were washed with 0.1× standard saline citrate (SSC)/0.1% SDS for 30 min at 65° C and exposed at 80° C for 2 d to X-OMAT film from Eastman Kodak Company (Rochester, NY). Relative intensities of the radioactive bands were quantitated by Bio-image analyzer (Fuji Film Ltd., Tokyo).

Enzyme-linked Immunosorbent Assay (ELISA) of MIF

The anti-rat MIF IgG polyclonal antibody dissolved in PBS (50 µl) was added to each well of a 96-well microtiter plate, which was then left for 30 min at room temperature. The plate was washed three times with distilled water. All wells were filled with PBS containing 0.5% BSA for blocking and left for 20 min at room temperature. After removal of the blocking solution, the samples were added in duplicate to individual wells and incubated for 1 h at room temperature. After the plate was washed three times with PBS containing 0.05% Tween 20 (washing buffer), 50 µl of biotin-conjugated anti-MIF antibody was added to each well. After incubation for 1 h at room temperature, the plate was again washed three times with the washing buffer. Avidin-conjugated horseradish peroxidase was added to each well, and the microtiter plate was again incubated for 15 min at room temperature. After washing three times, the substrate solution (10 ml) contained 8 mg of o-phenylenediamine and 4 µl of 30% H₂O₂ in citrate phosphate buffer (pH 5.0). Finally, the substrate solution (50 µl) was added to each well. After incubation for 20 min at room temperature, the reaction was terminated with 25 µl of 4 N sulfuric acid. The absorbance was measured at 492 nm by an ELISA plate reader (Model 3550; Bio-Rad). The detection limit in this system was 1.5 ng/ml.

ELISA of MIP-2/CINC-3

The level of MIP-2/CINC-3 in BAL fluids was measured and the LPS group and anti-MIF Ab group were compared with a specific rat MIP-2 ELISA kit using recombinant rat MIP-2/CINC-3 as a standard. Although three other subtypes of CINCs have been identified, such as CINC-1, CINC-2, and CINC-2, this system has no cross-reactivity for those (16). The detection limit in this system is reportedly 50 pg/ml.

Statistics

Data were analyzed by one-way analysis of variance (ANOVA), and individual group means were then compared with Students' t test. All values are expressed as mean ± SEM unless otherwise specified.

	RESULTS

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Effect of Anti-MIF Antibody on Lung Inflammation

Histology and MPO activity. No rats died in any treatment groups throughout the experimental period. There was a significant decrease in the counts of circulating blood thrombocytes at 24 h in the LPS group, but the leukocyte numbers did not decline (Table 1). This LPS-induced thrombocytopenia was significantly attenuated by pretreatment with the anti-MIF antibody. Light microscopic examination revealed a slight but significant degree of neutrophil accumulation in and around alveoli only in the rats that had received the nonimmunized rabbit IgG + LPS (Figures 1A and 1B). However, there were no serious lung injuries, such as pulmonary edema or hemorrhage, even in the LPS group, so that no significant difference was found in the W/D weight ratio between the control group and the LPS group (Table 1). The numbers of neutrophils observed in the lung tissues 4 and 24 h after administration of LPS were significantly larger than those of the control group (Figure 2A). When rats were pretreated with the anti-MIF antibody, the number of neutrophils significantly decreased compared with the LPS group (2.67 ± 0.33 versus 1.20 ± 0.09 cells/alveolus at 4 h, 2.45 ± 0.40 versus 1.38 ± 0.29 cells/alveolus at 24 h, p < 0.05). The MPO activity of lung tissue in the anti-MIF Ab group was also markedly lower than that of the LPS group at 4 and 24 h after administration of LPS (2.04 ± 0.27 versus 1.34 ± 0.11 OD/min at 4 h, 1.39 ± 0.15 versus 0.66 ± 0.24 OD/min at 24 h, p < 0.05) (Figure 2B). There was a significant and close correlation between the number of neutrophils observed and the activity of MPO in the lung tissues (r = 0.85 at 4 h, r = 0.93 at 24 h), indicating that two independent evaluations provided exactly the same results on neutrophil migration.

View larger version (96K): [in this window] [in a new window]   View larger version (87K): [in this window] [in a new window]   Figure 1.   Light microscopic examination. (A) Neutrophils significantly accumulated in and around alveoli in the LPS group; however, there were no serious lung injuries such as pulmonary edema and hemorrhage. (B) Pretreatment with anti-MIF antibody attenuated LPS-induced neutrophil accumulation.

View larger version (29K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 2.   Accumulation of neutrophils in LPS-injured lungs. Negative control rats received saline instead of LPS (n = 8). The significant attenuation of neutrophil migration by the anti-MIF antibody was proved by three independent examinations at 4 and 24 h after administration of LPS: nonimmunized rabbit IgG + LPS (n = 20), anti-MIF antibody + LPS (n = 20). (A) The number of neutrophils per alveolus, (B) activity of MPO in the lung, and (C ) the number of neutrophils in BAL fluid.

BAL findings. Although there were no significant differences among all groups either in the total cell number or in the albumin concentration in BAL fluid, the neutrophil differentiation ratio was significantly increased again only in the LPS group at 24 h after LPS injection (10.0 ± 2.7%) compared either with the control group (1.2 ± 0.4%, p < 0.05) or with the anti-MIF Ab group (2.0 ± 0.4%, p < 0.05) (Table 1, Figure 2C).

MIP-2/CINC-3 in BAL fluids. As is shown in Figure 3, the level of MIP-2 in BAL fluid was significantly lower in the anti-MIF Ab group than in the LPS group (253 ± 62 pg/ml versus 759 ± 101 pg/ml, n = 5 for each, p < 0.01).

View larger version (12K): [in this window] [in a new window]   Figure 3.   MIP-2/CINC-3 in BAL fluid. The MIP-2 levels in BAL fluids from the rats pretreated with anti-MIF antibody were significantly decreased at 4 h after commencement of injury (from 759 ± 101 pg/ml to 253 ± 62 pg/ml, p < 0.01). All values are expressed as mean ± SEM, with n = 5 in each group.

MIF in serum and BAL fluids. The level of MIF in serum was 14 ± 3 ng/ml in the control rats (n = 5). The serum level significantly and markedly increased at 4 h (121 ± 25 ng/ml, n = 8, p < 0.05) and also at 24 h (38 ± 9 ng/ml, n = 5, p < 0.05) in the rats that had been given LPS. However, we found no significant increases in the level of MIF in BAL fluids at the same periods (2.17 ± 0.48 ng/ml at 0 h, 3.20 ± 1.18 ng/ml at 4 h, and 3.08 ± 0.74 ng/ml at 24 h, respectively, not significant).

Immunohistochemical Localization and Identification of MIF in Lungs of Rats

Positive immunostaining for MIF was observed even in the control animals within the bronchial epithelial cells and in almost all alveolar macrophages (Figures 4A and 4B). There was a moderate increase in immunostaining in both cell types in the LPS-treated rats (Figures 4C and 4D). Northern blot analysis demonstrated prominent MIF expression in the lung tissue, and its mRNA levels clearly increased 24 h after LPS administration (Figure 5).

View larger version (148K): [in this window] [in a new window]   View larger version (141K): [in this window] [in a new window]   View larger version (147K): [in this window] [in a new window]   View larger version (145K): [in this window] [in a new window]   View larger version (144K): [in this window] [in a new window]   View larger version (143K): [in this window] [in a new window]   Figure 4.   Immunostaining for MIF in the lungs of rats (original magnification: ×200). Positive immunostaining for MIF was demonstrated even in the control rats within the bronchial epithelial cells (A) and alveolar macrophages (B). There was a moderate increase in immunostaining of bronchial epithelial cells (C ) and alveolar macrophages (D) at 24 h after LPS injection. The tissue specimen reacted with pre- immune rabbit IgG as a negative control; bronchial epithelial cells (E ), and alveolar macrophages (F  ).

View larger version (63K): [in this window] [in a new window]   Figure 5.   Northern blot analysis. Prominent expression of MIF was demonstrated in the lung tissues from control rats. Its mRNA level increased to 24 after LPS administration.

	DISCUSSION

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In this study, we demonstrated that pretreatment of rats with an anti-MIF antibody clearly attenuated the accumulation of neutrophils in the lungs 4 and 24 h after the administration of LPS. The significant attenuation of neutrophil accumulation caused by the anti-MIF antibody in this experiment was proved by three independent methods: histological examination of neutrophils in and around the alveoli, MPO activity of lung tissues, and quantification of neutrophils in BAL fluids. We also showed that MIF was present in bronchial epithelial cells and alveolar macrophages in the lungs and that MIF mRNA from lung tissues was certainly increased 24 h after the administration of LPS, although we could find increased levels of MIF only in serum, not in BAL fluids, in the rats that had been given LPS. Furthermore, we demonstrated that an increase in the level of MIP-2/CINC-3, a powerful neutrophil chemokine, in BAL fluids observed in the rats of the LPS group was significantly suppressed by pretreatment with the anti-MIF antibody. All these data indicate that MIF is implicated in the pathogenesis of LPS-induced neutrophil accumulation in the lungs and that the anti-MIF antibody has therapeutic potential for the treatment of acute lung injury, at least in part, by suppressing the level of a neutrophil chemokine in the lungs.

MIF was first described in 1966 as a lymphokine or a cytokine activity derived from activated T lymphocytes that had the ability to prevent the random migration of macrophages (1, 2). Since then, expression of MIF or MIF-like activity has been found at a variety of inflamed loci. However, the precise role of MIF in the immunologic response remained undefined until human MIF cDNA was cloned in 1989 (3). In a series of recent experiments using mice by Bucala and colleagues, MIF has been rediscovered as a pro-inflammatory cytokine that has the potential to override the anti-inflammatory action of glucocorticoids (5, 6). In sepsis, the level of MIF in blood increases, and administration of recombinant MIF enhances lethality. Conversely, pre-injection of an anti-MIF antibody could fully protect mice from lethal endotoxemia (4). Although it has been reported that the major sources of MIF secretion are the pituitary glands and macrophages as well as T lymphocytes (4, 6, 17), it is now known that the MIF gene is expressed in a wide variety of cells, tissues, and organs, including the brain, spleen, liver, muscle, and kidney (7, 11). We previously reported for the first time the crystal structure of MIF (18), and provided evidence that MIF is also present in the differentiating cells of the cornea and the skin (8, 9).

There has been, to our knowledge, only one report examining the presence of MIF in the lungs in experimentally induced endotoxemia (11). In this study, positive immunostaining for MIF was observed in untreated animals within the bronchial epithelium and in alveolar macrophages. The level of MIF mRNA increased markedly 24 h after LPS administration. These findings on the localization of MIF in the lungs agree with those previously reported in animals as well as in humans (11, 12). In this study, however, we could not detect a significant increase in the immunologic level of MIF in BAL fluids in the rats that had been given LPS, although marked elevations in the serum level were seen 4 and 24 h after LPS administration. Failure to recognize increased levels of MIF in BAL fluids in those rats may have occurred because the lung injury caused by LPS was not severe enough in this experiment. The model of LPS-induced acute lung injury in rats resembles ARDS in patients, and intraperitoneal administration of LPS generally results in neutrophil recruitment in the lungs and increased vascular permeability (19, 20). It is not clear why the dose of LPS used in this study did not cause severe lung damage, leading to a significant increase in the W/D weight ratio in the LPS-treated rats. In this study, we gave 7 mg/kg LPS to each rat, which is not considered to be a small quantity.

The mechanism by which the anti-MIF antibody attenuated neutrophil accumulation in the lungs was thought to be, at least in part, due to its suppressive effect on a putative neutrophil chemokine, MIP-2/CINC-3. In this experiment, the level of MIP-2/CINC-3 in BAL fluids was significantly lower in the anti-MIF Ab group than in the LPS group 4 h after LPS administration. It was recently reported that MIP-2/CINC-3 plays a significant role in the LPS-induced inflammatory response in rat lungs and is required for the full recruitment of neutrophils (21, 22). The peak level of MIP-2/CINC-3 in BAL fluids was observed 4 h after intratracheal instillation of LPS in these studies. It remains to be elucidated whether this suppressive effect of the anti-MIF antibody on a neutrophil chemokine is a direct action of the antibody itself on MIF as a pro- inflammatory cytokine, or an indirect effect potentiating the action of endogenous glucocorticoids, which MIF is reported to counter-regulate.

Despite long-term intensive research efforts, the etiology of ARDS remains uncertain and no specific therapy has proven effective in either preventing or reversing the underlying injury (23). A number of trials using antibodies or inhibitors against various stages of the inflammatory process have been experimentally attempted. They included neutrophil elastase inhibitor (24), antibodies to E- and L-selectin (25), an antibody to IL-8 (26), and an antibody to endotoxin (27). However, none of them has demonstrated any significant improvement in clinical trials. As an endogenous inhibitor of glucocorticoid action, MIF may modulate the delicate balance between the proinflammatory cytokines, such as TNF- and IL-8, and the anti-inflammatory effect of endogenous steroids in an early phase of ARDS. Since MIF is considered to be not merely one more cytokine because of its potential for overriding the actions of endogenous steroids, the findings of this study may lead to new anti-inflammatory therapy for ARDS. Further studies will be required to determine the effect of the anti-MIF antibody on severe lung injuries and, moreover, its possible beneficial effect when the antibody is given after the initiation of lung injury.