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Published ahead of print on March 30, 2006, doi:10.1165/rcmb.2005-0272OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 198-205, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0272OC

Role of Macrophage Migration Inhibitory Factor in Acute Lung Injury in Mice with Acute Pancreatitis Complicated by Endotoxemia

Naoyuki Matsuda, Jun Nishihira, Yoshika Takahashi, Osamu Kemmotsu and Yuichi Hattori

Department of Pharmacology, School of Medicine, University of Toyama, Toyama; Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, Sapporo; and GeneticLab Company, Limited, Sapporo, Japan

Correspondence and requests for reprints should be addressed to Yuichi Hattori, M.D., Ph.D., Department of Pharmacology, School of Medicine, University of Toyama, Toyama 930-0194, Japan. E-mail: yhattori{at}med.u-toyama.ac.jp


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Acute pancreatitis accompanied by a subsequent infectious attack can often lead to multisystem organ dysfunction, including acute lung injury (ALI), but the molecular mechanisms are poorly defined. In this study, we explored the role of the priming insult by induction of cerulein pancreatitis, which was followed by the second attack due to endotoxemia, in the development of ALI in mice. Experiments revealed that LPS injection in mice with acute pancreatitis caused the development of ALI, as indicated by blood-gas derangements, pulmonary vascular hyperpermeability, increased inflammatory cell counts in bronchoalveolar lavage, and histologic lung damage. This was associated with the pancreatitis-induced increase in expression of macrophage migration inhibitory factor (MIF) in the lungs, together with elevated expression of Toll-like receptor (TLR)-4, both of which were inhibited by administration of anti–protease-activated receptor (PAR)-2 antibody. Furthermore, anti-MIF antibody treatment suppressed the pancreatitis-induced elevation of TLR-4 pulmonary expression. Genetic removal of MIF from mice resulted in less development of ALI in the setting of acute pancreatitis complicated by endotoxemia. These findings demonstrate that activation of protease-activated receptor–2 with trypsin, which can be released after pancreatitis induction, positively regulates the transcript level of MIF, and increased MIF results in exaggerated pulmonary expression of TLR-4, leading to the development of ALI with a subsequent infectious attack. We thus suggest that interventions designed to modulate MIF may have therapeutic advantages in treating ALI in patients with acute pancreatitis complicated by bacterial infection.

Key Words: acute lung injury • acute pancreatitis • macrophage migration inhibitory factor • protease-activated receptor–2 • Toll-like receptor–4


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Acute pancreatitis is mild and self-limiting, for the most part, but patients may incidentally develop a severe disease that is characterized by local pancreatic necrosis, as well as systemic organ failure (1). Severe pancreatitis is often associated with bacterial infectious complications (2). Infectious complications can result in a poor prognosis, with hemodynamic instability and multiple organ failure leading to death in some cases. Endotoxin, which plays a key role in the initiation of septic shock caused by gram-negative bacteria, has been given considerable attention as a major contributor to pancreatitis-associated multiple distant organ injury (3). Acute lung injury (ALI) is one of the critical complications of severe acute pancreatitis (4), but the molecular mechanisms by which ALI develops during severe pancreatitis are still poorly understood.

Endotoxin, the LPS portion derived from the bacterial cell wall, activates systemic inflammatory responses through Toll-like receptor (TLR)-4 (5). Indeed, the demonstration that TLR-4–deficient mice exhibit hyporesponsiveness to LPS (6) suggests that TLR-4 is the gene product that regulates LPS response. Interestingly, recent work has shown that elastase, commonly found in extracellular fluids after acute pancreatitis (7, 8), triggers systemic inflammatory response syndrome in mice via stimulation of TLR-4 (9). More recently, it has been documented that the deficiency in TLR-4 can fully prevent LPS-induced aggravation of pancreatitis-associated ALI in mice (10). Therefore, TLR-4 appears to play an important role in the molecular basis for the pathophysiology of pancreatitis-associated ALI accompanied by bacterial infectious complications.

MIF was originally identified as a cytokine derived from activated T cells (11, 12). However, MIF is now considered to exert various biologic functions in macrophage activation (13–15). Moreover, MIF is thought to play a central role in exacerbation of inflammation and sepsis (16, 17). Importantly, a recent report has suggested that gene expression of TLR-4 in macrophages can be upregulated by MIF (18). Thus, hyporesponsiveness of MIF-deficient macrophages to LPS has been demonstrated by a marked reduction in the activity of NF-{kappa}B and the production of TNF-{alpha}, which is strictly associated with downregulation of TLR-4.

In the present study, we chose to use a mouse model with severe acute pancreatitis that was induced by cerulein, a decapeptide analog of the pancreatic secretagogue, cholecyctokinin, and was aggravated by a subsequent single intravenous injection of LPS. Injecting mice with cerulein leads to the development of mild edematous pancreatitis (19). A severe form of acute pancreatitis, characterized by local organ injury (pancreatic necrosis) and distinct organ damage or dysfunction, can be induced by a relatively minor secondary insult (administration of a small dose of LPS) (20–22). Because TLR-4 is a molecule of the LPS receptor complex necessary to transduct the signal of LPS into cells (23), we hypothesized that the regulation and function of TLR-4 may be critical in the onset and development of ALI on this two-hit model. Therefore, MIF, which is an important modulator of TLR-4, may be actually involved in the initiation of distinct organ injury, such as ALI, which stems from acute pancreatitis complicated by endotoxemia. Also, with the use of MIF-deficient gene knockout mice, it was of interest to better understand the molecular mechanisms by which inflammatory signals, including MIF-associated TLR-4 modulation, could influence ALI formation in the setting of acute pancreatitis complicated by endotoxemia.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Animals used in this study included MIF-deficient and wild-type (WT) BALB/c mice. MIF-deficient mice were established by targeted disruption of the MIF gene, as described previously (24), using a mouse strain onto a BALB/c background. All mice were kept on a 12:12-h light:dark cycle, with free access to food and water. The Hokkaido University School of Medicine Animal Care and Use Committee approved all animal procedures.

Experimental Model
Age-matched (8- to 12-wk-old) male mice received hourly subcutaneous injections of cerulein (20 µg/kg/h) for 3 h to induce pancreatitis. One hour after the last cerulein injection, a single injection of 4 mg/kg LPS (Escherichia coli 055:B5) was administered intravenously as the septic challenge. Saline was substituted for cerulein or LPS in control animals. Animals were killed 2 h after injection of LPS. Lung tissues were harvested, immediately frozen in liquid nitrogen, and stored at –80°C for future studies.

Wet-to-Dry Weight Ratio
Surgically removed lung tissues were blotted dry and weighed to determine the lung wet weight. The lung tissues were then wrapped loosely in aluminum foil, placed in a drying oven overnight, and weighed again for calculation of the wet-to-dry weight ratio (25, 26).

Pulmonary Microvascular Permeability
Pulmonary microvascular permeability was evaluated by quantitating the leakage of intravenously administered 125I-labeled albumin into the bronchoalveolar space (25, 26). The ratio of radioactivity between bronchoalveolar lavage fluid (BALF) and blood was calculated and expressed as the permeability index.

Cell Counting in BALF
The alterations in cells in BALF were examined in a separate group of experimental animals. To lavage the bronchoalveolar space, 2 ml PBS was instilled via the trachea and then gently withdrawn. The BALF was then centrifuged at 1,000 x g for 10 min to separate cells from the supernatant. Cells were resuspended in saline and counted with a hemacytometer. Slides were stained with Wright stain and cell differential was tabulated using light microscopy at x40 magnification.

Histologic Examination
To obtain lungs for routine histology, the trachea was cannulated and the lungs were gently inflation-fixed en bloc with 4% buffered formalin solution (2 ml). Inflation-fixed lungs were harvested, fixed, dehydrated, paraffin-embedded, and sliced into 4-µm-thick sections. Before the tissue sections were labeled, they were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The sections were stained with hematoxylin and eosin and examined using light microscopy (final magnification, x100).

Western Blot Analysis
Immunoblotting was performed as described in our previous report (27). Samples of tissue homogenate (5–20 µg protein) were run on a 7.5 or 12.5% SDS polyacrylamide gel and transferred to a polyvinylidine difluoride filter membrane. The membrane was then blotted with the indicated antibody and processed via chemiluminescence.

RNA Extraction and Northern Blot Analysis
Total RNA was extracted from lung tissues by the guanidinium thiocyanate-phenol-chloroform method with Isogen, used routinely in our laboratory (28). Northern blot was performed as previously described (27), with slight modifications. Briefly, 30-µg samples were electrophoresed on agarose/formaldehyde gels and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences Ltd, Little Chalfont, UK). The filter was baked, prehybridized, and hybridized with 32P-labeled cDNA probe. The mouse TLR-4 cDNA (accession no. U88880) was isolated from gel after electrophoretic separation of the products that were amplified by the PCR using two oligonucleotide primers (sense, 5'-CCAGAGTTTCCTGCAATGGATCAAGGAC-3'; and antisense, 3'-TCAGATAGATGTTGCTTCCTGCCAATTGC-5') (29). The intensity of hybridization was visualized by autography. Expression of mRNA was quantitated by counting the radioactivity using a bioimaging analyzer (Fujix BAS 2000; Fuji Photo Film, Tokyo, Japan) (28). Ethidium bromide staining was used as a control to verify gel loading, and expression of TLR-4 mRNA was normalized as the ratio of TLR-4 mRNA over 28 S ribosomal RNA.

Survival Studies in Acute Pancreatitis Complicated by Endotoxemia
Additional groups of mice, WT and MIF–/–, underwent induction of acute pancreatitis followed by administration of 4 mg/kg LPS, and were included in survival studies. WT mice were randomly divided into two groups. One group of WT mice received intravenous injections of anti-MIF antibody (20 mg/animal) immediately before the first cerulein injection, and the other group was given only saline injections. The animals were allowed free access to food and water, and survival was recorded for 2 d.

Statistical Analysis
The data are presented as mean ± SEM (n = 4–5). Data were analyzed using the Stat View II program (Abacus Concepts, Berkeley, CA). Statistical analysis was performed using Student's t test or a repeated-measures one-way ANOVA followed by Bonferroni's multiple comparison test, where appropriate; a P value less than 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Induction of Pancreatitis by Cerulein
Serum amylase and lipase levels were measured 4 h after the last dose of cerulein was injected. Induction of pancreatitis in cerulein-treated mice was evidenced by greatly elevated serum amylase (4,868 ± 446 IU/liter) and lipase (1,498 ± 40 IU/liter) compared with the levels in saline-treated control animals (375 ± 70 and 61 ± 9 IU/liter, respectively). LPS did not exacerbate the degree of pancreatitis in cerulein-treated mice. Thus, serum amylase and lipase levels were 5,880 ± 395 and 1,998 ± 138 IU/liter, respectively. Two hours after administration of LPS, these levels were essentially the same as those obtained in the pancreatitis animals treated with saline instead of LPS (5,938 ± 390 IU/liter for amylase; 1,880 ± 158 IU/liter for lipase).

Assessment of ALI in the Two-Hit Model
Table 1 summarizes the values for blood gases in mice after treatment with cerulein and/or LPS. When the animals were given cerulein or LPS alone, the blood gas parameters were not much different from the control values. However, mice who received both cerulein and LPS injection exhibited a severe hypoxemic condition, as indicated by markedly reduced arterial PO2 in comparison with control mice. Arterial PCO2 had a tendency to decrease when mice suffered from acute pancreatitis with endotoxemia, but this decrease was not statistically significant. A significant reduction in arterial blood pH and a greatly lowered value for base excess, indicating metabolic acidosis, were measured in mice with endotoxemic pancreatitis.


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  		TABLE 1. INFLUENCES OF ACUTE PANCREATITIS AND/OR ENDOTOXEMIA ON BLOOD GASES IN MICE

 
When lung microvascular permeability was assessed by the transpulmonary flux of radiolabeled albumin (Figure 1A), induction of acute pancreatitis with cerulein and intravenous LPS challenge alone caused only a 2.1- and 3.9-fold increase in lung permeability, respectively. The intravenous injection of LPS 1 h after the last dose of cerulein resulted in an 18.7-fold increase in lung permeability. The wet-to-dry lung weight ratios for each group were also measured for assessment of changes in lung vascular permeability (Figure 1B). The wet-to-dry lung weight ratios of mice treated with cerulein or LPS alone were slightly but significantly increased as compared with those of control mice. However, the endotoxemic pancreatitis group showed a much greater ratio than the control group.


Figure 1
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  		Figure 1. Influences of acute pancreatitis and/or endotoxemia on lung permeability, lung water, and BALF cells in mice. (A) Pulmonary transcapillary radiolabeled albumin transit was used to assess alterations in lung permeability. Intravenous injection of 10 µCi of 125I-labeled albumin was performed 30 min before the animals were killed. (B) Wet-to-dry (W/D) ratios of lungs harvested from the animals were determined to assess pulmonary edema. (C) After BALF was collected from lungs, total inflammatory cells were counted with a hemacytometer. All data are presented as means ± SEM; n = 4. *P < 0.05 compared with control; #P < 0.05 compared with acute pancreatitis alone.

 
As shown in Figure 1C, quantitation of inflammatory cells detected in BALF showed 2.9- and 6.7-fold increases in the number of cells recovered from mice with acute pancreatitis and endotoxemic mice, respectively, as compared with that seen in the lungs of control mice. As anticipated, intravascular challenge with LPS in mice with acute pancreatitis caused a more marked increase (22.4-fold) in the number of BALF inflammatory cells. Differential staining showed that acute pancreatitis followed by endotoxemia resulted in a notable rise in neutrophils in BAL cell contents as well as in macrophages (Table 2).


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  		TABLE 2. EFFECTS OF ACUTE PANCREATITIS AND/OR ENDOTOXEMIA ON CELL DIFFERENTIAL IN BRONCHOALVEOLAR LAVAGE FLUID OF MICE

 
Histologic examination of hematoxylin and eosin–stained sections of the lungs demonstrated that normal control mice had alveolar septae that were normal in appearance, with no intra-alveolar inflammation (Figure 2A). Lung histopathology of mice with induced pancreatitis showed mild edema with thickening of the alveolar septum, but inflammatory cell infiltrate was minimal (Figure 2B). Mice with induced pancreatitis that received LPS had massive cell inflammation, hemorrhage, enlargement of the alveolar airways, and diffuse septal edema (Figure 2C). The inflammatory cells were mainly composed of macrophages and were clumped within the alveolar air spaces.


Figure 2
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  		Figure 2. Hematoxylin and eosin–stained sections of lung tissues harvested from mice. Images represent (A) lung from a control mouse, (B) lung from a mouse with acute pancreatitis alone, and (C) lung from a mouse with acute pancreatitis followed by LPS treatment. Lung histology in acute pancreatitis and endotoxemia complications shows interstitial and intra-alveolar inflammatory infiltrate (arrows) and hemorrhage, as well as diffuse septal edema. Images are representative of at least three animals from each group. All images are at x100 magnification (scale bar = 100 µm).

 
Pulmonary Expression of Inflammatory Molecules in the Two-Hit Model
On Western blots, the 130-kD protein of inducible nitric oxide synthase (iNOS) was very weakly detectable in lungs from control mice (Figure 3A). Induction of acute pancreatitis or endotoxemia caused a modest but significant increase in the amount of iNOS protein in lungs. When mice with acute pancreatitis were simultaneously rendered endotoxemic by LPS, a much greater increase in the iNOS protein expression level in lungs was found.


Figure 3
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  		Figure 3. Influence of acute pancreatitis and/or endotoxemia on NF-{kappa}B activation and expression of {kappa}B-associated gene molecules in mouse lungs. Pulmonary expression levels of (A) iNOS and (B) ICAM-1 proteins in mice. The upper traces show typical Western blot, indicating marked increases in expression of a major band for each target molecule in acute pancreatitis followed by LPS. Actin served as loading control. (C) Mice were subjected to acute pancreatitis with and without endotoxemia before nuclear extracts were obtained from the lungs and processed for Western blot analysis; a typical Western blot, indicating a marked increase in p65 protein in acute pancreatitis followed by LPS, is shown in the upper panel. All data are presented as mean ± SEM; n = 5. *P < 0.05 compared with control; #P < 0.05 compared with acute pancreatitis alone.

 
Western blot analysis of intracellular adhesion molecule (ICAM)-1 protein revealed that its 90-kD protein expression level was 2- and 3.2-fold higher in lungs from mice with acute pancreatitis and from endotoxemic mice, respectively, than in control lungs (Figure 3B). This increase was much more marked in mice with acute pancreatitis complicated by endotoxemia. Thus, the ICAM-1 protein level was increased 8.6-fold by acute pancreatitis with LPS challenge.

The genes for the inflammatory molecules, including iNOS and ICAM-1, can be regulated by {kappa}B sites in the DNA, which bind NF-{kappa}B proteins (30–32). To examine the activation of NF-{kappa}B in mouse lungs, we measured NF-{kappa}B translocation to the nucleus with the antibody to an NF-{kappa}B subunit of p65. As presented in Figure 3C, translocation of NF-{kappa}B to the nucleus was increased 2.8-fold in lungs from mice with acute pancreatitis, and 5.1-fold in lungs from mice with endotoxemia. However, LPS treatment of mice with acute pancreatitis resulted in a 16.7-fold increase in NF-{kappa}B translocation to the nucleus in lungs.

Altered Levels of MIF and TLR-4 by Acute Pancreatitis
Plasma MIF levels were markedly elevated, from 14.9 ± 3.5 to 104.5 ± 7.3 µg/ml (P < 0.001) at 4 h after the first injection of cerulein. In addition, pulmonary expression of MIF protein was increased 4.7-fold by induction of acute pancreatitis (Figure 4). The pancreatitis-induced increase in pulmonary MIF expression was strongly inhibited by the antibody to protease-activated receptor (PAR)–2, a receptor that is activated by trypsin and tryptase (33) (100 µg/animal, sc-9278 P; Santa Cruz Biotechnology, Santa Cruz, CA).


Figure 4
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  		Figure 4. Immunoblot analysis of MIF protein in lungs from control mice and mice with acute pancreatitis. When anti–PAR-2 antibody (100 µg/animal) was used, it was given intravenously immediately before the first cerulein injection. (A) Representative Western blot indicating an increase in expression of a 12.5-kD band in acute pancreatitis. (B) Bar graph summarizing the immunoblot data. The data are presented as mean ± SEM; n = 5. *P < 0.05 compared with control mice; #P < 0.05 compared with acute pancreatitis alone.

 
Northern blot analysis of total RNA prepared from mouse lungs revealed the presence of two TLR-4 transcripts at 9.5 and 5 kb (Figure 5A). Following induction of acute pancreatitis by cerulein injection, the two transcript levels were markedly increased (Figure 5B). The anti–PAR-2 antibody showed a strong inhibition of the pancreatitis-induced increase in TLR-4 mRNA expression. Furthermore, the increased gene expression level of TLR-4 was nearly completely prevented by the antibody against MIF (20 µg/animal, produced by J.N.). Immunologic detection of TLR-4 was performed using an anti-goat TLR-4 polyclonal antibody (Santa Cruz Biotechnology), which recognized a 100-kD band in mouse lungs (Figure 5C). Immunodetectable TLR-4 was found at significantly higher levels after induction of acute pancreatitis. Densitometric quantification of the signal showed that the TLR-4 protein level was 5.1-fold higher in lungs of mice with acute pancreatitis compared with that in lungs of control mice. Treatment with the anti-MIF antibody or the anti–PAR-2 antibody greatly suppressed the pancreatitis-induced increase in pulmonary expression of TLR-4 at protein levels.


Figure 5
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  		Figure 5. Gene and protein expression of TLR-4 in lungs from mice. When anti-MIF antibody (20 µg/animal) or anti–PAR-2 antibody (100 µg/animal) was used, they were given intravenously immediately before the first cerulein injection. (A) Representative autograph of Northern blot analysis of TLR-4 mRNA expression. TLR-4 mRNA was detected as the major two bands of 5 kb and 9.5 kb by using a 32P-labeled cDNA fragment (+1925 to +2684) of mouse TLR-4 as a probe. Ethidium bromide staining was used as a control for gel loading (lower panel). (B) Bar graph summarizing the Northern blot data. Densitometric measurement of TLR-4 mRNA was normalized as the ratio of its mRNA level over 28 S level. (C) Blocking effects of anti-MIF antibody and anti–PAR-2 antibody on the increased pulmonary expression level of TLR-4 by acute pancreatitis. (D) Lack of increasing effect of acute pancreatitis on TLR-4 expression in lungs from MIF knockout (MIF–/–) mice. The upper traces in (C) and (D) show typical Western blots. Actin served as loading control. All data are presented as mean ± SEM; n = 5. *P < 0.05 compared with control (WT); #P < 0.05 compared with WT with acute pancreatitis alone.

 
Lack of ALI Due to Pancreatitis Followed by Endotoxemia in MIF-Deficient Mice
No MIF was detected in the plasma of MIF-deficient (MIF–/–) mice. MIF–/– mutant mice responded normally to cerulein. At 1 h after the last dose of cerulein was injected into MIF–/– mice, serum amylase and lipase levels were increased from 448 ± 66 and 53 ± 14 IU/liter to 4,232 ± 699 and 1,574 ± 115 IU/liter, respectively.

Pulmonary expression of TLR-4 was slightly but significantly lower in MIF–/– mice than in WT mice (Figure 5D). Whereas induction of acute pancreatitis with cerulein resulted in a 5.2-fold increase in TLR-4 expression in lungs from WT mice, the pancreatitis-induced increment of pulmonary TLR-4 expression was much less pronounced in MIF–/– mice.

When blood gases were measured in arterial blood samples from MIF–/– mice, arterial pH, PCO2, and PO2 were not significantly altered by induction of acute pancreatitis followed by endotoxemia (Table 3). Although the attack of both acute pancreatitis and endotoxemia caused a significant decrease from baseline for base excess in MIF–/– mice, this decrease was much less pronounced than that seen in WT mice (see Table 1). Thus, pancreatitis/endotoxemia-induced blood-gas derangements were evidently eliminated in MIF–/– mice.


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  		TABLE 3. VAGUE EFFECT OF ACUTE PANCREATITIS AND ENDOTOXEMIA ON BLOOD GASES IN MIF+/– MICE

 
When assessed by the transpulmonary influx of radiolabeled albumin, the calculated lung permeability index was increased from 0.010 ± 0.002 to 0.036 ± 0.007 in MIF–/– mice that had an attack of both acute pancreatitis and endotoxemia. However, this increase in lung permeability was much smaller than that observed in WT mice when they suffered from both acute pancreatitis and endotoxemia (see Figure 1A).

From the start, there was a significant increase in the number of inflammatory cells from the BALF of MIF–/– mice (15 ± 2 x 105 cells/ml; P < 0.05) compared with that seen in the lungs of WT mice (6 ± 1 x 105 cells/ml). Nevertheless, induction of acute pancreatitis with endotoxemia failed to result in a prominent increase in the number of inflammatory cells from the BALF of MIF–/– mice (32 ± 3 x 105 cells/ml). This was in sharp contrast to the marked increase in the number of BALF inflammatory cells in WT mice with acute pancreatitis complicated by endotoxemia (132 ± 19 x 105 cells/ml).

Histologic examination of the lungs demonstrated that MIF–/– mice had normal lung architecture despite induction of acute pancreatitis followed by endotoxemia (Figure 6). MIF–/– mice originally showed a moderate inflammatory cell infiltrate along the alveolar septae, but cellular inflammation was no longer accelerated, even if acute pancreatitis and endotoxemia were induced.


Figure 6
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  		Figure 6. Hematoxylin and eosin–stained sections of lung tissues harvested from MIF knockout (MIF–/–) mice. Images represent (A) lung from an untreated MIF–/– mouse, (B) lung from a MIF–/– mouse with acute pancreatitis alone, and (C) lung from a MIF–/– mouse with acute pancreatitis followed by LPS treatment. Lung histology shows that MIF deficiency prevents lung injury caused by acute pancreatitis and endotoxemia complications, as indicated by less inflammatory infiltrate (arrows). Images are representative of at least three animals from each group. All images are at 100x magnification (scale bar = 100 µm).

 
Survival after Induction of Acute Pancreatitis/Endotoxemia
All animals survived after induction of acute pancreatitis or endotoxemia alone; however, mortality was 90% at 2 d after acute pancreatitis/endotoxemia induction in WT mice (Figure 7). Mortality was drastically improved (10%) with a single administration of anti-MIF antibody in the group of WT mice that had both acute pancreatitis and endotoxemia. All MIF–/– mice survived through 2 d after induction of acute pancreatitis complicated by endotoxemia.


Figure 7
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  		Figure 7. Kaplan–Meier survival curves for WT and MIF–/– mice after induction of acute pancreatitis followed by endotoxemia with LPS. The survival curves were obtained over a 2-d interval. Anti-MIF antibody (20 µg/animal) was given to mice intravenously immediately before the first cerulein injection. Ten mice were used for each group.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
According to the two-hit hypothesis (34, 35), an initial overactive insult, such as acute pancreatitis, somehow primes the inflammatory response. Recovery is possible if no further insult occurs. Bacterial infection as a relatively minor secondary attack will, however, lead to an exaggerated secondary inflammatory response, which may be fatal. In this study, we found that the onset of acute pancreatitis caused by cerulein administration, without the secondary attack by endotoxin challenge, did not induce remote organ injury, such as ALI, in mice. We demonstrated that administering a small dose of endotoxin (4 mg/kg) to the mouse in the setting of cerulein acute pancreatitis resulted in increased lung inflammation at an early time point (6 h from the initiation of pancreatitis). The given dose of endotoxin alone had no significantly injurious effect on lungs. In mice without a primary injury, 10 mg/kg endotoxin could induce ALI at 10 h after application (25). We thus believe that the two-hit model is appropriate for gaining insight into the molecular mechanism underlying the role of the priming insult followed by the secondary attack in the development of remote organ injury.

NF-{kappa}B is usually retained in an inactive form in the cytoplasm by a member of the family of inhibitory {kappa}B proteins; most commonly, inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) (36). Upon stimulation, I{kappa}B{alpha} is phosphorylated, ubiquitinated, and degraded, freeing NF-{kappa}B to the nucleus, where it regulates gene activity. We observed that NF-{kappa}B nuclear translocation was increased in lungs from mice with acute pancreatitis complicated by endotoxemia to a much greater extent than in those from mice with each disease alone. NF-{kappa}B is considered to play a key role in transcription of multiple genes involved in the inflammatory response that leads to organ dysfunction and failure (37). Indeed, we have recently demonstrated that in vivo transfection of NF-{kappa}B decoy deoxynucleotides can prevent sepsis-induced ALI in mice by suppressing the transactivation of many {kappa}B-associated inflammatory genes (25, 26). We thus interpret our present observations to indicate that NF-{kappa}B activation, which was abnormally elevated in lung tissues, caused excessive expression of inflammatory genes and contributed to the development of ALI when endotoxin invaded in the setting of acute pancreatitis. Consistent with this interpretation is the finding that acute pancreatitis complicated by endotoxemia resulted in extraordinary increases in pulmonary expression of iNOS and ICAM-1, both of which are inflammatory molecules from {kappa}B-associated genes (30–32); these increase occurred despite only a modest increase in expression of these molecules in acute pancreatitis or endotoxemia alone.

LPS stimulates cells by interaction with CD-14 in the context of TLR (38). Then, TLR activation leads to NF-{kappa}B nuclear translocation through degradation of I{kappa}B{alpha} and subsequent release of NF-{kappa}B. Compelling genetic data now suggest that TLR-4, acting in conjunction with the secreted protein MD-2, is the sole receptor for LPS (5, 23). We found that pulmonary expression of TLR-4 was greatly upregulated at both gene and protein levels after induction of acute pancreatitis. Due to this overexpression of TLR-4, lungs in the setting of acute pancreatitis may respond excessively to LPS, even from a small dose, which would result in increased NF-{kappa}B activation and subsequent overproduction of many inflammatory molecules, leading to the development of ALI. Further support for this idea can be provided by a recent report (10) showing that LPS fails to aggravate pancreatitis-associated ALI in TLR-deficient mice.

In the present study, treatment with anti–PAR-2 antibody fully prevented the pancreatitis-induced increase in the gene and protein expression levels of TLR-4 in lungs. This suggests that activation of PAR-2 is involved in upregulation of TLR-4 in lungs of mice with acute pancreatitis. However, it should be emphasized that treatment with anti–PAR-2 antibody also blocked the pancreatitis-induced increase in pulmonary MIF expression. Increased MIF levels in serum, ascitic fluids, and organs have been documented in patients with acute pancreatitis and in experimental acute pancreatitis animal models (39, 40). Furthermore, anti-MIF antibody treatment was found to result in a strong suppression of increased pulmonary expression of TLR-4 in the setting of acute pancreatitis. This finding is in accord with the previous results obtained in macrophages showing upregulation by MIF of TLR-4 expression (18). PAR-2 can be activated by trypsin and tryptase, as well as by some coagulation factors, but not by thrombin (33). Therefore, it would be reasonable to conclude that stimulation of PAR-2 with trypsin, which can be released in large quantities after induction of acute pancreatitis, upregulates the transcript level of MIF, and increased MIF results in exaggerated expression of TLR-4 in lungs.

When the cerulein model of acute pancreatitis was characterized by marked elevations of serum amylase and lipase levels, the severity of cerulein-induced acute pancreatitis was identical in WT and MIF–/– mice. Nevertheless, acute pancreatitis failed to increase TLR-4 expression in lungs of MIF–/– mice. This is more compelling evidence that increased expression of MIF levels leads to an elevation of TLR-4 expression in lungs in the setting of acute pancreatitis. When compared with WT mice, even if induction of acute pancreatitis was followed by LPS administration, MIF–/– mice minimally exhibited blood gas exchange impairment, accelerated pulmonary vascular permeability, and increased BALF inflammatory cell counts or histologic damage in lungs. Thus, ALI developed in acute pancreatitis followed by endotoxemia was evidently mitigated in MIF–/– mice. The lack of harmful effects of endotoxemic pancreatitis on lungs in MIF–/– mice is most likely the result of no induction of TLR-4 overexpression in lungs. The results of the study employing MIF–/– mice provide sound evidence that MIF is a strong inducer of TLR-4, and this pathway is important in the development of ALI in the setting of acute pancreatitis complicated by endotoxemia. Our present data are in good agreement with earlier reports showing TLR-4 modulation by MIF (18, 41). In addition, recent work suggests that MIF plays a crucial role in the alveolar inflammation associated with acute respiratory distress syndrome (42).

During acute pancreatitis complicated by endotoxemia, deficiency of MIF evidently decreased mortality. Although mortality in WT mice was 90% for the 2-d observation period, all MIF–/– mice survived. Based on the present results from the molecular experiments, we interpret this finding to indicate that the presence of endogenous MIF, which is abundantly expressed in pancreatitis, induces upregulation of TLR-4 expression. It may also be involved in the development of mutisystem injury, including ALI, and the death of WT mice that received LPS injection following acute pancreatitis. We also observed that treatment with anti-MIF antibody drastically improved survival in WT mice after induction of pancreatitis followed by LPS administration. Thus, we demonstrate the possibility that this strategy could provide an excellent survival benefit in acute pancreatitis complicated by secondary bacterial infection. Because survival benefit remains the major goal of preclinical testing, this information provides valuable insight into the introduction of interventions designed for MIF as a target for treatment of patients suffering from severe acute pancreatits with bacterial infectious complications.

It has been demonstrated that recombinant mouse MIF greatly enhances lethality when coinjected with LPS, and that polyclonal antibodies against the recombinant protein provide mice with full protection from LPS-induced lethal septic shock (16). However, previous work has shown that there is no significant difference between WT and MIF–/– mice in the survival rate after intraperitoneal injection of LPS (12 mg/kg) (24). Thus, it has been concluded that MIF is not crucial for LPS-induced immune responses leading to lethal shock. From the present results, it is likely that the presence of acute pancreatitis as a preceding priming effect is important in the exaggeration of responses of the organs to secondary insult endotoxin. The exaggerated endotoxin-induced response is associated with upregulation of TLR-4 expression due to pancreatitis-induced MIF overexpression. Accordingly, if no priming episode inducing overproduction of MIF precedes endotoxemia, the role of MIF in the inflammatory injury process in simple endotoxemia would be minimal.

It should be kept in mind that the MIF-mediated inflammatory responses may not always depend upon TLR-4 signaling. Thus, it has been demonstrated that MIF may play a crucial role in the development of dextran sulfate sodium–induced colitis independently of the TLR-4 signaling pathway (43). MIF can induce cytosolic phospholipase A2 (44), which has been linked to the pathobiology of ALI from sepsis (45). Therefore, the possibility exists that several kinds of lipid mediators, such as platelet-activating factor and other eicosanoids, may be involved as the potential additional pathopysiologic mechanisms by which MIF plays a key role in the development of ALI in acute pancreatitis with endotoxemia.

In the present study, we delineated the molecular mechanism underlying the development of ALI in the setting of acute pancreatitis complicated by endotoxemia. To our knowledge, this is the first report clarifying the significance of acute pancreatitis as a preceding priming effect, and endotoxemia as a following secondary insult, in the developmental process of remote organ injury. The MIF–/– mouse model was an appropriate model to verify that PAR-2–activated elevations of MIF in lungs led to the development of ALI through overexpression of TLR-4 in cerulein pancreatitis followed by LPS administration. The results from this study demonstrate that the absence of MIF expression plays a preventive role in the development of ALI in the setting of acute pancreatitis with endotoxemic complications. Our data thus indicate that therapy with intervention designed to modulate MIF in patients with acute pancreatitis complicated by bacterial infection may prevent the development of multisystem injury, including ALI, thus minimizing the morbidity and mortality associated with such severe pancreatitis.


   Acknowledgments
 
The authors thank Prof. Toshihiko Iwanaga and Dr. Hiroo Teramae for assistance with histologic techniques. They are also grateful to Mami Fujinaga, Somako Tone, and Mitsue Azuma for their skillful technical assistance, to Mari Tsuchihashi and Megumi Matsui for expert secretarial assistance, and to Lesley D. Riley for proofreading.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research and for Exploratory Research from the Ministry of Education, Science, Sports, and Culture of Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0272OC on March 30, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 18, 2005

Accepted in final form February 2, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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