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Published as doi: 10.1096/fj.08-124248.
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(The FASEB Journal. 2009;23:1262-1271.)
© 2009 FASEB

Macrophage migration inhibitory factor is critical to interleukin-5-driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection

Elizabeth S. Magalhães*,1, Claudia N. Paiva*,1, Heitor S. P. Souza{dagger}, Alexandre S. Pyrrho{ddagger}, Diego Mourão-Sá*, Rodrigo T. Figueiredo*, Adriana Vieira-de-Abreu, Helio S. Dutra§, Mariana S. Silveira||, Maria Ignez C. Gaspar-Elsas#, Pedro Xavier-Elsas*, Patrícia T. Bozza and Marcelo T. Bozza*,2

* Departamento de Imunologia, Instituto de Microbiologia,

{dagger} Departamento de Clínica Médica Faculdade de Medicina,

{ddagger} Departamento de Farmácia, Instituto de Biologia,

§ Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, and

|| Laboratório de Neurogênese, Instituto de Biofísca, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and

Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, and

# Departamento de Pediatria, Instituto Fernandes Figueira, Fiocruz, Rio de Janeiro, Brazil

2Correspondence: Departamento de Imunologia, Instituto de Microbiologia, CCS Bloco I, UFRJ. Avenida Carlos Chagas Filho, 373 Cidade Universitária, Rio de Janeiro, RJ, 21941–902 Brazil. E-mail: mbozza{at}micro.ufrj.br; mtbozza{at}gmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF-deficient (Mif–/–) mice had similar numbers of adult worms, eggs, and granulomas compared to wild-type mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif–/– mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif–/– mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif–/– mice chronically infected with S. mansoni compared to wild type. Mif–/– had impaired eosinophilopoiesis in response to interleukin (IL)-5 and addition of rMIF to bone marrow cultures from IL-5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL-5-supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z-VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL-5-driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.—Magalhães, E. S., Paiva, C. N., Souza, H. S. P., Pyrrho, A. S., Mourão-Sá, D., Figueiredo, R. T., Vieira-de-Abreu, A., Dutra, H. S., Silveira, M. S., Gaspar-Elsas, M. I. C., Xavier-Elsas, P., Bozza, P. T., Bozza, M. T. Macrophage migration inhibitory factor is critical to IL-5-driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection.


Key Words: Th2 response • apoptosis • inflammation • granuloma • MIF


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MACROPHAGE MIGRATION INHIBITORY factor (MIF) is a critical mediator of the innate and acquired immune response, exhibiting several proinflammatory functions (1) and antiapoptotic activities (2 3 4) , and participating in the host response to bacterial and parasitic infections (5 6 7 8) as well as in the pathogenesis of many autoimmune and inflammatory diseases (5 , 9 , 10) . Recently, a role for MIF in atopic diseases emerged from a number of studies. Asthmatic patients have increased concentrations of MIF in the bronchoalveolar lavage fluids (11) , sputum, and sera (12) . Induction of MIF mRNA and protein has been observed in activated Th2 cells (13) , while eosinophils have preformed MIF and are able to secrete high quantities of it on stimulation (11) . Several studies demonstrated that MIF deficiency affects Th2 pathologies, reducing mucus secretion, airway hyper-responsiveness (AHR) and prominently, eosinophilic inflammation, in animal models of allergic rhinitis and asthma (14 15 16 17 18 19) . In parasitic diseases in which a Th2 response predominate, such as the response to helminthic infection by Taenia crassiceps and Schistosoma japonicum, an inefficient control of the adult forms has been observed in the absence of MIF (8 , 20) .

Eosinophils constitute a minor multifunctional leukocyte subpopulation involved in initiation and propagation of diverse inflammatory responses (21) and antigenic presentation to T cells (19 , 22) . Blood and tissue eosinophilia occurs in atopic and parasitic diseases, fueled by extended eosinophil life span and increased bone marrow eosinophilopoiesis. In asthma, products released by eosinophils can cause the bronchoconstriction and AHR (23) , and a fundamental role for these cells in asthma was recently confirmed in eosinophil-deficient mouse lineages (24 , 25) . Eosinophils are also considered important against several parasitic infections, particularly against helminths (21 , 26) . In Schistosoma mansoni infection, eosinophils constitute a prominent cell type in granulomas, and there is increased eosinophilopoiesis on infection (27) , a process dependent on the interleukin (IL)-5 secreted during Th2 response to eggs (28 , 29) . Mice deficient in IL-5, which have decreased eosinophilopoiesis, present reduced liver granuloma sizes with decreased numbers of eosinophils on S. mansoni infection (30) . In fact, eosinophils have been found to kill the S. mansoni parasite in vitro (26) and to invade dying and sick schistosomes in vivo (29) , suggesting that they might be an important defense mechanism against the parasite. However, two mouse lineages deficient in eosinophils were recently studied on S. mansoni infection and found to have no gross alterations in worm burden, granuloma formation, or liver fibrosis (31) . Though the role of eosinophils as a defense mechanism against S. mansoni infection remains to be elucidated, their increased bone marrow differentiation and tissue infiltration during Th2 responses seem to be regularly observed.

We have previously observed a marked decrease in bone marrow eosinophilia of Mif–/– mice on allergic airway provocation (16) . Faced with the multiple evidence of reduced eosinophilia in the absence of MIF (15 , 16 , 18 , 19) , we set out to investigate the role of MIF in bone marrow eosinophilopoiesis in vitro and in vivo, and also, how it affects tissue eosinophilia. To this later purpose, we studied S. mansoni infection, focusing on granuloma formation. Herein, we report that MIF is required as a survival factor for IL-5-driven differentiation of eosinophils and for eosinophil accumulation in granulomas on S. mansoni infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
We utilized 7- to 8-wk-old female Mif–/– mice and respective controls, all on the C57Bl/6–129/Sv F2 background (5) . IL-5-transgenic BALB/c mice were obtained from Drs. Sandra Perez and Peter Weller (Beth Israel, Harvard Medical School, Boston, MA, USA). Mice were kept at constant temperature (25°C) with free access to chow and water in a room with a 12 h light-dark cycle. The experiments were performed accordingly to guidelines of the Institutional Animal Welfare Committee.

S. mansoni infection
Wild-type and Mif–/– mice were infected with 50 cercariae of the BH strain of S. mansoni by the cutaneous route. Animals from all groups (≥5/group) were killed under anesthesia on the 55th day of infection. During this time, they were maintained in controlled temperature and light conditions, fed a balanced diet, and given sterile water ad libitum. In migration studies, we isolated eosinophils from C57BL/6 mice that were infected with 100 cercariae and sacrificed 80 days after infection.

Parasitological parameters
Hepatic and intestinal tissues were maintained in 4% KOH at room temperature for ~12 h, followed by 1 h of incubation at 37°C. Results were expressed as the number of eggs per parasite pair per gram of tissue. Counts were done in triplicate. Perfusion of mesenteric vessels was performed by opening the abdomen and chest under deep anesthesia, dissecting the portal vein, and introducing a catheter in the thoracic aorta or the left heart ventricle. The numbers and sexes of the adult worms were determined.

Histopathology and morphometric analysis
Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Sections of 5 µm were stained with hematoxylin-eosin, picrosirius red, and Masson (32) . Quantitative analysis of tissue sections and of captured images was carried out using a computer-assisted image analyser (Image-Pro Plus Version 4.1 for Windows; Media Cybernetics, LP, Silver Spring, MD, USA). One observer who was unaware of the experimental setting examined all tissue sections in a random fashion. Tissue sections were completely surveyed for granulomas, and digital photographs were obtained under light microscopy at x400 magnification. To estimate the size of granulomas, we measured their individual areas using the measurement tool of the image analyzer.

Quantification of cytokines
Cytokine concentrations in plasma were measured using commercially available ELISA kits for IL-5 and IL-13 (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol.

Measurement of total IgE
Blood samples were collected and serum was obtained and stored at –20°C until measurement of total IgE by ELISA (BD Pharmingen).

Eosinophil-enriched suspensions
Livers from S. mansoni-infected mice were homogenized, and intact granulomas were allowed to sediment. After multiple washes with RPMI 1640 medium, granulomas were transferred to culture bottles and left overnight at 37°C in 5% CO2/95% air to allow cell migration to culture medium. Medium was collected and centrifuged. Supernatants were collected and constituted the conditioned granuloma medium (CGM) used as a chemoattractant. Pellets were suspended in medium, and cells were counted. Cytospin smears were prepared and stained with Diff-Quick (Baxter Health Corp., McGraw Park, IL, USA) to determine the percentage of eosinophils in suspensions. The nonadherent fraction was composed by ~90% eosinophils. Eosinophil-enriched preparations were also obtained from IL-5 transgenic bone marrows through Percoll 72–65% discontinuous gradient, yielding a 94–95% pure population of eosinophils.

Transwell migration assay
One hundred microliters of 1% FCS-RPMI 1640 (migration medium) containing 105 to 2 x 105 cells from eosinophil-enriched preparations was placed in the upper chamber of Costar 24-well 6.5-mm transwell plates, with 5 µm pore size (Costar, Cambridge, MA). Chemoattractants were diluted in 600 µl migration medium and placed in the lower chamber of the transwell plates. Cells were blocked by addition of CXCR2 (SB225002, 1 µM) or CXCR4 antagonists (AMD3100, 100 ng/ml). Plates were incubated at 37°C in 5% CO2/95% air for 4 h, and then cells in the lower chamber were collected. The lower face of the inserts was washed with cold RPMI medium to remove the cells that remained attached to the bottom. The total suspension from the lower chamber was centrifuged and suspended in 100 µl. Cells were counted in Neubauer chambers, and cytospin smears were stained with Diff-Quick to determine the number of migrated eosinophils.

Bone marrow eosinophilia
Wild-type and Mif–/– mice were killed, and the femurs were removed. The bone marrow cavity was flushed with RPMI plus 1% FCS (HyClone, Logan, UT, USA). The cells were cytocentrifuged and eosinophil peroxidase-positive (EPO+) cells were counted.

Bone marrow cell cultures
Single-cell suspensions were prepared from bone marrow of wild-type and Mif–/– mice. The erythrocytes were removed by lysis using NH4Cl, and cells were plated. Liquid bone-marrow cultures (106 cells in a 1-ml volume, in a 24-well cluster) were seeded in RPMI 1640 medium with 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin and were maintained at 37°C in 5% CO2/95% air for 7 days. Recombinant mouse IL-5 (Pharmingen) or recombinant mouse MIF was added to the cultures in the first day of culture at the indicated concentrations. The frequency of EPO+ cells was determined in cytocentrifuge smears considering only viable cells. EPO+ cell fragments were not counted as eosinophil precursors or mature cells. In some experiments, the frequency of mature eosinophils was determined from smears stained with May-Grünwald Giemsa. The number of macrophages with at least one EPO+ cell fragment in the cytoplasm was determined by counting 300 macrophages. The addition of 30 µM of z-VAD (Enzyme System Products, Dublin, CA, USA) or 50 ng/ml of recombinant MIF (kindly provided by Christine Metz, The Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhasset, NY, USA) were performed in the first day of cell culture.

Statistical analysis
Statistical analysis was performed with Prism computer software (GraphPad Software, Inc., San Diego, CA, USA). Statistical differences among the experimental groups were evaluated by analysis of variance with Newman-Keuls correction or with the t test. Values are expressed as means ± SE. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MIF contributes to tissue eosinophilia in S. mansoni infection
The infection of mice and humans with S. mansoni causes a robust type 2 immune response characterized by the differentiation of naive CD4+ cells into Th2 cells and by the increased numbers of eosinophils in blood, tissues, and bone marrow (33) . To investigate the role of MIF in tissue eosinophilia, we infected Mif–/– and wild-type controls via percutaneous route with 50 cercariae and analyzed their livers and intestines 55 days after infection. Adult forms and eggs present in the liver and intestine were counted 55 days postinfection. Similar numbers of adult forms and eggs were found in wild-type and Mif–/– mice (Fig. 1A, B ), thus indicating that the lack of MIF did not interfere with the control of the infection. The presence of S. mansoni eggs in the tissues caused the formation of granulomas, characterized by the presence of macrophages, lymphocytes, and eosinophils (34) . The histopathological analysis of liver and intestine revealed a similar number of granulomas in wild-type and Mif–/– mice (Fig. 1C ). This result is compatible with the similar number of eggs found in both mouse strains (Fig. 1A ). The sizes of the granulomas both in livers and intestines were strikingly reduced in Mif–/– compared to wild-type mice (Fig. 2A, C ). The number of eosinophils in the granulomas of Mif–/– mice was also significantly reduced compared to wild-type mice (Fig. 2B, C ), which could account for the reduction in the sizes of granulomas. Nevertheless, the structure of tissue granulomas was preserved in Mif–/– mice. Collagen content of granulomas, as assessed by picrosirius red and Masson’s collagen staining, was similar among wild-type and Mif–/– mice (Fig. 2D ). Similar collagen deposition was also found in intestines (data not shown).


Figure 1
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  		Figure 1. Absence of MIF does not alter the course of S. mansoni infection. Mice were infected with 50 S. mansoni cercariae through a percutaneous route, and livers and intestines were collected 55 days postinfection. A) Eggs per gram of tissue (isolated from livers and intestines as described in Materials and Methods). B) Adult worms. C) Granulomas per square millimeter of slide section. Data represent means ± SE; 3–5 infected wild-type or Mif–/– mice were used as individual tissue donors. *P ≤ 0.05.


Figure 2
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  		Figure 2. Size and cellular composition of liver and intestine granulomas developed on S. mansoni infection are dependent on MIF. Slide sections from infected mice were examined under light microscope, and images were captured and analyzed as described in Materials and Methods. A) Individual granuloma area, calculated from images of 10–20 granulomas acquired for each individual mouse. B) Eosinophils per individual granuloma (n=3–5 mice/group). Data represent means ± SE. *P ≤ 0.05. C) Representative granulomas in liver and intestine sections from wild-type (WT) and Mif–/– mice (H&E; x400). D) Liver collagen stained with Sirius red (top panels; x100) or tricolor Masson staining (bottom panels; x400).

Th2 response to S. mansoni infection is not altered in Mif–/– mice
Impairment of the Th2 polarization can inhibit eosinophilopoiesis and reduce the size of granulomas on S. mansoni infection, such as occurs in IL-5 or in concomitant IL-13/IL4 deficiencies (30 , 35) . Therefore, we tested whether lack of MIF could alter Th2 polarization, and thus impair eosinophil-dependent inflammation. S. mansoni-infected wild-type and Mif–/– mice displayed similar plasma concentrations of IL-5 (Fig. 3A ) and IL-13 (Fig. 3B ). Moreover, absence of MIF did not impair the secretion of IgE on S. mansoni infection, and plasma IgE was even greater in infected Mif–/– mice than in wild-type controls (Fig. 3C ). These results indicate that MIF is not essential to Th2 differentiation induced by S. mansoni infection.


Figure 3
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  		Figure 3. MIF does not alter Th2 response to S. mansoni infection. A) IL-5 (n=6 mice/group). B) IL-13 (n=3–5 mice/group). C) Total IgE (n=8–9 mice/group). Data represent means ± SE; values per milliliter of plasma from infected WT and Mif–/– mice. *P ≤ 0.05.

MIF attracts eosinophils
As liver granulomas from S. mansoni-infected Mif–/– had smaller numbers of eosinophils than those from wild-type controls, and MIF is known to exert chemoattraction over neutrophils, monocytes, and T lymphocytes (36) , we tested whether MIF could also be a chemoattractor to eosinophils. Eosinophil-enriched cell suspensions were prepared from liver granulomas and submitted to transwell assay against eotaxin (100 ng/ml), conditioned granuloma medium (CGM) or recombinant MIF (rMIF; 100 ng/ml). Eotaxin and CGM attracted large numbers of eosinophils (Fig. 4 , top panel). rMIF was also able to attract eosinophils, and preblockage of cells with either CXCR2 or CXCR4 antagonists abolished this effect. A similar phenomenon was observed when eosinophil-enriched suspensions were prepared from the bone marrow of IL-5 transgenic mice and submitted to transwell assay against rMIF (Fig. 4 , bottom panel). These results suggest that MIF is a chemoattractor to eosinophils.


Figure 4
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  		Figure 4. MIF chemoattracts eosinophils. Eosinophil-enriched suspensions containing 105 cells, obtained from the livers of 4 pooled S. mansoni-infected C57BL/6 mice (90 days postinfection; top panel), and 2 x 105 cells obtained from the bone marrows of 3 individual IL-5 transgenic mice (bottom panel) were either left untouched (ct, control treatment) or preincubated with CXCR2 (SB225002, 1µM) and CXCR4 antagonists (AMD3100, 100 ng/ml) and left to migrate in transwell plates against rMIF, eotaxin, and conditioned granuloma medium (CGM) for 4 h at 37°C. Data represent means ± SE; values indicate migrated mature eosinophils found in 3–4 wells. *P ≤ 0.05.

Optimal bone marrow eosinophilopoiesis requires MIF
The increased production of eosinophils in the bone marrow is critical to seed tissues during helminthic infections and depends largely on the IL-5 produced during a Th2 response (28) . As tissue eosinophilia is reduced in infected Mif–/– mice, we tested whether MIF would be required to allow the increase in bone marrow eosinophilopoiesis induced on S. mansoni infection. No significant differences were observed in the percentages of EPO+ cells in freshly obtained bone marrow from Mif–/– versus wild-type mice (Fig. 5A ). Infection with S. mansoni caused a massive increase of EPO+ cells in the bone marrow of wild-type mice. Mif–/– mice infected with S. mansoni had significantly lower percentages of eosinophils among bone marrow cells when compared to wild types, indicating that MIF is required for optimal eosinophilopoiesis.


Figure 5
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  		Figure 5. MIF is required for IL-5-driven eosinophilopoiesis. A) Percentage of EPO+ cells in fresh bone marrow from S. mansoni-infected and noninfected (NI) control mice, collected 55 days postinfection (n=8–9 mice/group). B) EPO+ cells per culture of bone marrow cells from wild-type (WT) and Mif–/– donors cultivated in individual duplicates for 7 days in the presence of various concentrations of rIL-5 (n=3–5). C) EPO+ cells per culture of bone marrow cells from WT and Mif–/– donors cultivated in individual duplicates for 7 days in the presence of 50 ng/ml rMIF (n=3–5). D) Percentage of mature eosinophils in individual bone marrow cultures from IL-5-transgenic mice that were cultivated with 100 ng/ml rMIF (May-Grünwald Giemsa stained smears). Data represent means± SE. *P ≤ 0.05.

IL-5 regulates the terminal differentiation of committed eosinophil precursors and mice deficient in IL-5 are unable to produce increased numbers of eosinophils during allergic inflammation or helminthic infection (21) . To test the hypothesis that MIF participates in eosinophil terminal differentiation, we used an in vitro system of eosinophil maturation induced by IL-5 (37) . Bone marrow cells from Mif–/– and wild-type mice were cultured for 7 days in the presence of murine recombinant IL-5. Viable EPO+ polymorphonuclear cells (corresponding to immature and mature eosinophils) accumulated in IL-5-stimulated cultures from wild-type, but not from Mif–/– (Fig. 5B ). The increase in the numbers of EPO+ cells among wild-type bone marrow cells after 7 days of culture was dependent on the IL-5 concentration, but bone marrow cells from Mif–/– mice failed to differentiate in EPO+ cells even in the presence of high IL-5 concentrations. The replacement of the Mif–/– cell cultures with rMIF (50 ng/ml) fully restored the ability of IL-5 (1 ng/ml) to promote accumulation of EPO+ cells to the numbers achieved in the wild type (Fig. 5C ). However, in the absence of IL-5, rMIF was unable to induce eosinophil differentiation (cultures remained at 1–2% EPO+ cells). Also, in IL-5 transgenic bone marrow cultures, which present a very active eosinophilopoiesis with high percentage of mature eosinophils by day 7, addition of 100 ng/ml rMIF was still able to produce a 3 fold increase in this percentage (Fig. 5D ), resulting in increased production of mature eosinophils. These results indicate that MIF acts as a cofactor necessary to allow optimal IL-5-driven eosinophilopoiesis both in vitro and in vivo.

After 7 days in the presence of IL-5, wild-type bone marrow cell cultures contained viable EPO+ cells, while Mif–/– cells had several small EPO+ bodies inside mononuclear cells and very few EPO+ cells (Fig. 6A ). The number of mononuclear cells with at least one EPO+ body inside was significantly increased in the cell cultures of Mif–/– mice compared to wild-type controls (Fig. 6B ). These results lead us to hypothesize that in the absence of MIF most of the eosinophil precursors would not be able to survive the terminal differentiation process induced by IL-5. To verify this hypothesis, we used the pancaspase inhibitor zVAD in the bone marrow cell cultures. The treatment of Mif–/– cell cultures with zVAD restored the number of EPO+ cells accumulated in the presence of IL-5 to the level observed in wild-type controls, indicating that in the absence of MIF the eosinophil precursors are more prone to die by apoptosis (Fig. 6C ). Addition of zVAD did not influence the IL-5-driven differentiation of EPO+ cells in bone marrow wild-type cultures, and z-VAD did not alter the course of differentiation in cultures not supplemented with IL-5 (both wild-type and Mif–/– cultures had 1–2% EPO+ cells after 7 days). Thus, MIF is essential to protect murine eosinophils from programmed cell death during terminal differentiation induced by IL-5 in culture.


Figure 6
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  		Figure 6. MIF is an antiapoptotic factor in eosinophil differentiation. Bone marrow cultures of WT or Mif–/– mice were cultivated in individual mouse duplicates for 7 days in the presence of 1 ng/ml of IL-5. A) Images show brown staining for EPO+ cells (eosinophils and precursors) or EPO+ staining in corpses inside monocytes, more frequent in cultures from Mif–/– mice (arrows).Top panels: x40; bottom panels: x100. B) Monocytes with EPO+ corpses were counted among total bone marrow cells from WT or Mif–/– (n=5 mice/group). C) ZVAD (30 µg) was added to IL-5-supplemented bone marrow cultures and percentage of EPO+ cells was calculated (n=3 mice/group). Data represent means ± SE. *P ≤ 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have demonstrated that MIF affects the production of eosinophils without interfering with the Th2 response. Mice deficient in MIF were unable to fully augment the numbers of eosinophils in response to IL-5 in vitro or to S. mansoni infection in vivo. MIF was essential to tissue eosinophilia but not to the control of S. mansoni adult forms or eggs. In this regard, Mif–/– mice had smaller granulomas with fewer eosinophils during S. mansoni infection. However, the plasma concentrations of IL-5, IL-13, and IgE were independent of MIF, indicating that it is not critical to Th2 polarization. Addition of rMIF restored the ability to differentiate eosinophils in Mif–/– bone marrow cultures, demonstrating that Mif–/– does not have a secondary defect on eosinophilopoiesis. Also, the inhibition of caspase activation with zVAD fully restored the number of EPO+ cells induced by IL-5 to the level observed in wild-type bone marrow cultures, thus indicating that in the absence of MIF, the eosinophil precursors are dying by apoptosis. Our results demonstrate a previously unrecognized role for MIF as a survival factor for eosinophils.

Similar numbers of S. mansoni adult forms and eggs were found in wild-type and Mif–/– mice, thus indicating that the lack of MIF does not interfere with the control of the infection. In contrast, Mif–/– mice have previously been shown to poorly control infection with the murine cisticercosis helminthic parasite T. crassiceps (8) . It is possible that the different outcomes of helminthic infections in the absence of MIF reflect the susceptibility of these parasites to the different defense mechanisms in which MIF interferes, which are currently poorly established. In fact, in mice treated with neutralizing antibodies against MIF, which have an increased parasite burden and fertility on S. japonicum infection compared to control antibody-treated animals (20) , this effect was only observed when treatment started late after infection, but not before infection, suggesting that there could be a time window during which secretion of MIF favors infection control.

Eosinophils can cause parasite damage in S. mansoni and other worm infections in vitro (26) and in vivo (34) . Nevertheless, mice selectively deficient in eosinophils (31) or in IL-5, which causes a profound reduction of eosinophils, control parasite burden as efficiently as wild-type mice (28 , 30) . In agreement with these data, we have shown here that S. mansoni infection is efficiently controlled despite the reduced numbers of eosinophils in Mif–/– mice. The role of eosinophils in controlling S. mansoni infection, if there is one, remains to be demonstrated. Contrasted with S. mansoni infection, Mif–/– mice are more susceptible to T. crassiceps than wild types, but have increased percentages of eosinophils infiltrating their peritonea (8) . In the face of the impaired eosinophilopoiesis we have found in vitro and in vivo (on both allergic airway provocation and S. mansoni infection), it is surprising how Mif–/– mice can still react with tissue eosinophilia to T. crassiceps infection. This increased eosinophilia could reflect either the existence of a MIF-independent eosinophil differentiation pathway evoked by T. crassiceps; the expression of a MIF homologue such as that of other helminthes (38 , 39) , allowing bone marrow eosinophilopoiesis; or, still, preferential homing and survival of eosinophils in the peritoneum, as previously proposed by others (40) .

The formation of granulomas around eggs depends on Th2 responses, as demonstrated in IL-4/IL-13 (35) , IL-10/IL-4 (41) , and IL-4{alpha}R deficient mice (42) , in which development of Th1-polarized responses to eggs leads to abnormal granulomas and liver damage. Here, we observed a striking reduction in the size of granulomas, likely due to the reduction in the number of infiltrating leukocytes, especially eosinophils. The reduction in size of the liver and intestine granulomas in S. mansoni-infected Mif–/– mice does not seem to be the consequence of an impaired Th2 response, since infected Mif–/– mice have high plasma concentrations of IL-5 and IL-13, as well as increased plasma levels of IgE. These results confirm and extend previous findings from our group and others indicating that MIF is not required to the Th2 differentiation during allergic inflammation and helminthic infection (16 , 8) . Also, these results indicate that the control exerted by MIF on the size of egg granulomas is not related to the disturbance of the Th2 response.

Secretion of IL-13 (43 , 35) and tissue eosinophilia have been indicated as participating in the fibrosis and consequent portal hypertension that causes morbidity during the chronic phase of the infection. Our analysis of collagen deposition in chronically S. mansoni-infected mice revealed that MIF is not essential for liver fibrosis induced by Th2 inflammation, since despite the reduced eosinophil infiltration, there was a similar collagen deposition in Mif–/– mice compared to wild types. Though a recent study demonstrated a critical role for eosinophils on collagen deposition and tissue remodeling induced by OVA immunization and challenge using mice that completely lack eosinophils (25) , a collagen deposition similar to wild-type controls was found in the livers of these eosinophil-ablated mice when they were infected with S. mansoni (31) . As production of IL-13, on the other hand, has been confirmed by several studies as a main factor inducing fibrosis (30 , 35) , we believe that the similar plasma levels of IL-13 we have found in infected Mif–/– and wild-type mice can account for their similar collagen deposition.

The reduced number of eosinophils in both the lungs of Mif–/– animals sensitized and challenged with OVA (16 , 8) and in the granulomas of S. mansoni- infected mice was associated with reduced production of eosinophils in the bone marrow. Though MIF action as a survival factor for eosinophils in the bone marrow probably fosters the tissue eosinophilia, it is likely that MIF also acts downstream to promote eosinophil extended survival in peripheral tissues. Currently, GM-CSF, IL-5, IL-3, IL-15, and eotaxin (44 45 46 47 48) are known survival factors to eosinophils in tissues, probably acting after transmigration and contributing to inflammation. Nonexclusively, MIF could participate in eosinophil recruitment to tissues. In this regard, a homologue of human MIF from Brugia malayi induces eosinophil inflammation (38) , suggesting that MIF could be an eosinophil recruiter. Herein, we demonstrated that MIF is required to tissue eosinophilia in S. mansoni infection and that it is capable of attracting eosinophils in transwell assays, a migration blocked with either CXCR2 or CXCR4 antagonists. In fact, the SDF receptor CXCR4 and the MIP-2/KC receptor CXCR2 were recently shown to be chemokine receptors for MIF (36) . As eosinophils express CXCR4 and are attracted by its ligand SDF-1 as efficiently as by eotaxin (49) , while CXCR4 blockade inhibits eosinophilia in both asthma (50) and S. mansoni granulomas (51) , these results suggest a role for CXCR4 ligands in eosinophil recruitment to lungs and liver. Also, mouse eosinophils express CXCR2 (52) , and their migration toward KC has been attributed to CXCR2, a receptor known to be upregulated in the presence of high IL-5 levels in humans (53) . Though we cannot currently discard the possibility that a contaminating cell type attracted by MIF is responsible for eosinophil recruitment, such as reported for neutrophils in human eosinophil preparations (54) , the putative role for MIF as both a recruiter and a survival factor for eosinophils, acting after transmigration and promoting the persistence of eosinophilic inflammation, demands systematic studies, especially due to its potential as a therapeutic target in asthma.

The lower production of eosinophils in bone marrow cultures from Mif–/– mice occurred in the presence of high IL-5 levels in the plasma. Moreover, the increase in bone marrow eosinophilopoiesis on S. mansoni infection, a process previously demonstrated to be dependent on IL-5 (28) , could not be fully achieved in the absence of MIF. Consistently, addition of MIF was capable of increasing the production of eosinophils in already high-producing IL-5 transgenic bone marrow cell cultures. Together, these results indicate that MIF provides an optimal IL-5-driven eosinophilopoiesis. Considering the ability of eosinophils to produce MIF (11) , it is conceivable that the IL-5 dependent effects observed in vitro might be due to an autocrine/paracrine effect of MIF release by eosinophil precursors. However, the mechanisms by which MIF increases IL-5-driven eosinophilopoiesis are still to be established. The presence of several EPO+ bodies inside mononuclear cells in the bone marrow cell cultures suggested that in the absence of MIF eosinophil precursors died by apoptosis and were removed by macrophages. In fact, prior studies demonstrated that MIF has antiapoptotic activities on fibroblasts, macrophages, and granulocytes (2 3 4) . In macrophages, MIF inhibits p53-mediated apoptosis through the activation of ERK1/ERK2, PLA2, COX2, and PGE2 (4) . The antiapoptotic mechanism of MIF on neutrophils involves the delayed cleavage of the proapoptotic molecules Bid and Bax, as well as the blockade of cytochrome c and Smac release from mitochondria, consequently inhibiting the caspase-3 activation (3) . We also demonstrated that zVAD, a pancaspase inhibitor, or rMIF fully restored the generation of eosinophils induced by IL-5 in bone marrow cell cultures of Mif–/– to the levels of wild type, thus indicating that MIF acted as a survival factor for eosinophil precursors during eosinophilopoiesis.

In conclusion, these studies demonstrate that MIF is a potent inflammatory mediator that plays an essential role on eosinophil biology. The reduced granulomas in Mif–/– mice on S. mansoni infection occurred regardless of a robust antigen-driven immune response, thus indicating that MIF is downstream of these events. Moreover, our results implicate MIF as a survival factor for eosinophil precursors, providing critical signals to the terminal differentiation process induced by IL-5. Together, these results demonstrate a previously unrecognized role of MIF on eosinophil generation and tissue accumulation.


   ACKNOWLEDGMENTS
 
We thank Sandra Perez and Peter Weller (Beth Israel, Harvard Medical School, Boston, MA, USA) for IL-5 transgenic mice. This work was supported by Conselho de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação José Bonifácio (FuJB), Howard Hughes Medical Institute (HHMI), and Programa de Núcleos de Excelência (Pronex).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication November 5, 2008. Accepted for publication November 26, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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