By
From the * Department of Immunology and Infectious Diseases, Harvard School of Public Health, and
the Ina Sue Perlmutter Laboratory, Children's Hospital, Harvard Medical School, Boston,
Massachusetts 02115
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Abstract |
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To study the biologic role of migration inhibitory factor (MIF), a pleiotropic cytokine, we
generated a mouse strain lacking MIF by gene targeting in embryonic stem cells. Analysis of
the role of MIF during sepsis showed that MIF/
mice were resistant to the lethal effects of
high dose bacterial lipopolysaccharide (LPS), or Staphylococcus aureus enterotoxin B (SEB) with
D-galactosamine and had lower plasma levels of tumor necrosis factor
(TNF-
) than did
wild-type mice, but normal levels of interleukin (IL)-6 and IL-10. When stimulated with LPS and interferon
, macrophages from MIF
/
mice showed diminished production of TNF-
,
normal IL-6 and IL-12, and increased production of nitric oxide. MIF
/
animals cleared
gram-negative bacteria Pseudomonas aeruginosa instilled into the trachea better than did wild-type mice and had diminished neutrophil accumulation in their bronchoalveolar fluid compared to the wild-type mice. Thioglycollate elicited peritoneal exudates in uninfected MIF
/
mice, but showed normal neutrophil accumulation. Finally, the findings of enhanced resistance
to P. aeruginosa and resistance to endotoxin-induced lethal shock suggest that the counteraction or neutralization of MIF may serve as an adjunct therapy in sepsis.
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Introduction |
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Macrophage migration inhibitory factor (MIF)1 is a pleiotropic cytokine released by macrophages, T cells, and the pituitary gland during inflammatory responses (1, 2). It has been shown to act as a proinflammatory cytokine, playing a major role in endotoxin shock (3) and counter-regulating the antiinflammatory effects of dexamethasone (4). Antibodies to MIF diminish the manifestations of autoimmunity in certain experimental models (5, 6). Furthermore, recent studies have shown that MIF enhances resistance to the pathogen Leishmania major (7, 8). Its ubiquitous expression (9) and developmental regulation (10, 11) suggest that MIF might have functions beyond the immune system.
To study the role of MIF, we generated a mouse strain
lacking MIF by gene targeting in embryonic stem cells and
analyzed mechanisms of sepsis using these MIF/
mice.
Sepsis triggered by gram-negative and gram-positive bacterial infection is a major cause of death of hospitalized patients (12). Endotoxin induces MIF release from macrophages and pituitary cells, as well as in vivo. MIF has been
shown to be released in large quantities and found in the
serum of mice after challenge with LPS (3, 13). A critical
role of MIF in endotoxemia was suggested by the observation that recombinant MIF enhanced LPS-induced lethality, whereas anti-MIF antibodies had a protective effect (3).
In this study, we describe the generation of MIF
/
mice
and characterize the specific role of MIF in sepsis.
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Materials and Methods |
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Targeting Vector Construction and Generation of MIF/
Mice.
LPS-induced Shock and Cytokine Measurement.
8-12-wk-old, sex-matched MIF+/+, MIF+/Shock Induced with LPS, TNF-, and Staphylococcus Enterotoxin B
with D-galactosamine.
Macrophage Cultures.
Macrophages elicited for 4 d with thioglycollate or resident macrophages were obtained by peritoneal lavage using 10 ml of PBS. 106 cells in 1 ml of RPMI/10% FCS supplemented with glutamine,Cytokine and Nitric Oxide Assays.
TNF-Lung Infection with Pseudomonas aeruginosa.
The mucoid P. aeruginosa strain A01 was used and maintenance of stocks and methods used were as previously described (16). Mice matched in age and gender (8-10 wk) were anesthetized with Ketamine (90 mg/kg) and Xylazine (10 mg/kg) (both from Sigma Chemical Co.). P. aeruginosa (3.5 × 106), freshly grown in tryptic soy broth for 18 h, were instilled into the trachea in a volume of 50 µl. The lung clearance of P. aeruginosa was measured by killing mice immediately and at 6 and 24 h after P. aeruginosa instillation. Lungs were excised and homogenized in 3 ml of ice-cold sterile distilled water. Homogenates were diluted appropriately in sterile PBS, cultured overnight in brain-heart infusion agar plates, and the number of CFU was determined. Results are expressed as CFU 6 h/CFU 0 h and CFU 24 h/CFU 0 h. To assess microvascular injury and neutrophil accumulation, 100 µl of Evans blue (6.25 mg/ml) was administered intravenously 2 h after P. aeruginosa instillation, and bronchoalveolar lavage (BAL) × 1 ml was performed 6 h after bacterial instillation. The recovered BAL fluid was assessed for total cell counts using a standard hemocytometer, and differential cell counts were determined from Dif-Quick- stained (Dade Diagnostics) cytospin preparations. Permeability changes were determined by comparing the leakage of Evans blue into the BAL fluid to the amount remaining in the plasma (17). ![]() |
Results and Discussion |
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The mouse
MIF gene was disrupted by replacing part of exons 2 and 3 with a neor cassette (Fig. 1 A). The targeting vector was electroporated into J1 ES cells and G418-FIAU-resistant colonies
were isolated. The average frequency of homologous recombination was about 1 in 30 resistant colonies. Correctly targeted ES cells were used to generate chimeric animals by
injection into C57BL/6 blastocysts. Highly chimeric animals
transmitted the mutated allele through the germline, and
homozygous mice were generated by intercrosses of heterozygous mice (Fig. 1 B). Northern blot analysis from liver RNA of LPS-treated animals confirmed that the gene disruption created a null mutation (Fig. 1 C). ELISA of serum
from LPS-treated animals further confirmed the absence of
MIF protein in the MIF/
mice (Fig. 1 D). Of the 218 animals obtained from heterozygous matings, 16% were homozygous for the null allele. The newborn MIF
/
mice developed normally in size and behavior and were fertile. The
litter size of heterozygous and homozygous matings were
normal. Both gross examination and histopathological analysis
of several organs (kidney, liver, spleen, adrenal, thymus,
lungs, heart, brain, and intestine) of MIF
/
mice revealed
no abnormalities. Flow cytometric analysis of splenocytes and
thymocytes of MIF
/
mice demonstrated normal lymphocyte populations (data not shown).
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To
analyze the role of MIF in endotoxemia, MIF/
, MIF+/
,
and wild-type mice were injected intraperitoneally with a
high dose of LPS (25 mg/kg). MIF deficiency conferred a
remarkable resistance to the lethal effects of LPS (Fig. 2 A).
However, MIF
/
mice still exhibited signs of endotoxemia a few h after LPS treatment, including piloerection,
shivering, and lethargy, although these signs appeared
milder than in control mice. Since a cascade of inflammatory mediators triggered by LPS is important in the pathogenesis of endotoxic shock (18, 19), we measured cytokine
levels in the plasma of MIF
/
mice compared with wild-type 90 min after LPS challenge. There was a 50% reduction in the plasma levels of TNF-
but similar levels of IL-6
and IL-10 (Fig. 2 B). The observed resistance to LPS could
be partially due to the diminished TNF-
production secondary to lack of MIF (13); to an enhanced antiinflammatory effect of stress-induced steroids no longer counter-regulated by MIF (4); or to possible other proinflammatory
properties of MIF which have yet to be determined.
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Macrophages were studied as they are critically involved in
the pathogenesis of endotoxemia (20). Furthermore, MIF
and TNF- have been shown to work in an autocrine/
paracrine fashion; i.e., MIF increases macrophage TNF-
,
which in turn increases MIF release (13). To study the effect of MIF deficiency on macrophage activation, thioglycollate-elicited peritoneal macrophages were stimulated with LPS and IFN-
for 6, 12, 18, and 24 h, and the supernatants were analyzed. Macrophages from MIF
/
mice
showed a marked decrease in TNF-
(P < 0.001; Fig. 3 A). However, MIF
/
macrophages were able to produce
IL-12 and IL-6 (Fig. 3, B and C). These results show that
MIF is required for the optimal production of TNF-
during endotoxemia. The lowered plasma levels of TNF-
are
probably at least partially due to diminished production by macrophages.
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It has been shown that recombinant MIF enhances NO
production in vitro by macrophages stimulated with IFN-
(21). Furthermore, attenuated Salmonella transfected with
the MIF gene alone, or in combination with TNF-
or
IFN-
administered orally to susceptible mice, reduced L. major infection and enhanced NO production (7). Surprisingly, there was a significant increase (P < 0.005) in NO in
the MIF
/
macrophages stimulated with LPS and IFN-
(Fig. 3 D). These results suggest that endogenous macrophage MIF either dampens NO production by these cells
or increases its turnover.
To further analyze the role of MIF
in lethal endotoxemia, we took advantage of mouse models
that have been developed to closely mimic the high sensitivity of humans to the toxic effects of bacterial products.
Hepatocytes from mice sensitized with D-galactosamine have a transcriptional arrest and become highly susceptible
to the cytotoxic action of TNF- (22, 23). The reduction
of TNF-
found in MIF
/
mice suggested that MIF acted
upstream from TNF-
but might still be involved in the
toxic effect of TNF-
. However, injection of a lethal dose
of TNF-
(1 µg/mouse) with D-galactosamine killed all
five wild-type and all five MIF
/
mice (Table I), indicating that MIF is not required for the lethal effects of TNF-
.
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Although MIF/
mice were resistant to a high dose of
LPS (Fig. 2 A), they were susceptible to a combination of
low-dose LPS and D-galactosamine; all five mice in each
group died (Table I). Indeed, it has been shown that the
mechanisms leading to death are different in models using
high or low doses of LPS (24).
Further studies showed that MIF/
mice were resistant
to the lethal effects of another bacterial product, SEB, with
D-galactosamine. All five wild-type mice died from SEB injected intraperitoneally whereas all five MIF
/
mice lived.
SEB acts as a superantigen for T cells, resulting in the release of inflammatory cytokines involved in toxic shock (25). Sera taken 90 min after SEB showed 65% less TNF-
in MIF
/
than in wild-type mice, 140 ± 25 and 396 ± 89 pg/ml, respectively (± SE, P < 0.05). These results indicate
that MIF plays a critical role in superantigen-induced toxic
shock in which T cells play a major role.
It is interesting to note that the phenotype of intracellular adhesion molecule (ICAM)-1-deficient mice is similar,
showing resistance to high doses of LPS as well as low
doses of SEB with D-galactosamine, and susceptibility to a
combination of a low dose of LPS and D-galactosamine
(26). ICAM-1-deficient mice have reduced transendothelial leukocyte migration but normal TNF- levels in response to LPS. However, the mechanisms of action in this
phenotype are different for MIF
/
mice, as the latter have
reduced TNF-
production and no impairment of leukocyte migration to the peritoneum elicited by thioglycollate (mean neutrophil count after 4 h was 6.71 ± 2.23 × 106
and 11.9 ± 2.15 × 106 in wild-type (n = 3) and MIF
/
(n = 4) mice, respectively, P < 0.1).
To determine the
role of MIF in host defense to gram-negative bacteria, P. aeruginosa were instilled intratracheally into MIF/
and
wild-type mice. The MIF
/
mice efficiently cleared bacteria from the lungs 24 h after infection, having almost 3 log fewer bacteria than heterozygous or wild-type controls
(MIF
/
9.3 ± 4.0 × 103, versus combined MIF+/+ and
MIF+/
2.1 ± 0.9 × 106, P < 0.03, Fig. 4 A). Of interest,
there was a significant decrease in neutrophils in the BAL at
6 h in the MIF
/
mice compared to controls, 55.3 ± 7.6 × 105 versus 95.9 ± 13.5 × 105, (n = 6, P < 0.04); Fig. 4 B. This is consistent with the report that anti-MIF antibody
diminishes LPS-induced neutrophil migration to the lungs
and BAL fluid as well as the level of macrophage protein 2, a powerful neutrophil chemokine (27).
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Inflammation has usually been thought to be a necessary
part of the host's defense against microorganisms, something the body must accept to successfully defend itself.
However, the findings reported here, along with the enhanced bacterial clearing and diminished inflammation in
mice lacking the CD14 receptor for endotoxin (28), suggest that host defense is more efficient without certain LPS-induced inflammatory cytokines. Indeed, IL-1 and TNF-
can enhance the growth and invasiveness of pathogenic gram-negative bacteria (29, 30). Moreover, MIF has been
found in the alveolar airspaces of patients with adult respiratory distress syndrome (ARDS), and increases the secretion of proinflammatory cytokines from alveolar cells (31).
We have initiated studies on MIF/
mice to determine
the effect of MIF on other infectious agents. In a preliminary experiment using the gram-positive bacteria L. monocytogenes, MIF
/
mice were not more susceptible than
wild-type controls. Furthermore, peritoneal macrophages
obtained from MIF
/
mice and stimulated with IFN-
and LPS were able to kill intracellular L. monocytogenes as
well as wild-type macrophages (data not shown). In contrast, MIF
/
mice were more susceptible than wild-type
mice to the intracellular parasite L. major. Lymph node cells
from infected MIF
/
mice showed higher IL-4 production after antigen stimulation than did those from wild-type
animals, suggesting that MIF plays a role in Th1/Th2
balance (our unpublished results).
Taken together, our results show that MIF plays a critical
role in endotoxic shock and SEB toxicity without hampering the ability of mice to clear gram-negative or -positive
infections. Indeed, the increased resistance to the gram-negative bacterial product LPS, as well as the enhanced
ability to clear P. aeruginosa infections in the lungs in MIF/
mice, indicates that neutralization or counteraction of MIF
might constitute an adjunct therapy for the treatment of
sepsis. Further studies with this animal model should clarify
the role of MIF in immunity, inflammation, and other biologic functions.
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Footnotes |
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Address correspondence to John R. David, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Phone: 617-432-1201; Fax: 617-738-4914; E-mail: jdavid{at}hsph.harvard.edu
Received for publication 22 September 1998 and in revised form 5 November 1998.
Marcelo Bozza's present address is Laboratorio de Farmacologia Aplicada, Far Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.We thank Cox Terhorst, John Samuelson, Willy Piessens, and Roberta David for their critical reading of the manuscript.
This work was supported in part by National Institutes of Health grant AI22532-13. M. Bozza was partially supported by a grant from CNPq, Brazil.
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