t1 dm is an autoimmune - mediated disease characterized by selective destruction of insulin - producing pancreatic -cells , resulting in the need for lifelong administration of exogenous insulin for patient survival , and represents 510% of all cases of diabetes [ 2 , 3 ] .
the pathological condition begins with an autoimmune inflammatory process known as insulitis , which leads to leucocyte infiltration into the pancreatic islets of langerhans , resulting in the autoimmune destruction of pancreatic -cells .
thus , the onset of t1 dm is the result of the recognition of self - antigens due to molecular mimicry against infection with various viruses such as coxsackie virus , -cell cytotoxicity , or -cell cytolysis [ 57 ] .
consequently , molecules such as preinsulin , insulin , glutamate decarboxylase-65 ( gad-65 ) , and islet antigen 2 become recognized as self - antigens .
different types of innate immune cells such as t cells , eosinophils , macrophages ( m ) , and dendritic cells ( dc ) as well as proinflammatory cytokines and chemokines are present during insulitis [ 4 , 9 ] . the relationship between t1 dm and high levels of inflammatory cytokines such as interleukin- ( il- ) 1 [ 1014 ] , interferon- ( ifn- )  , tumor necrosis factor- ( tnf- )  , il-12 [ 17 , 18 ] , and macrophage migration inhibitory factor ( mif ) [ 1924 ] has been widely recognized .
mif is a pleiotropic cytokine produced during the immune response by activated t cells , m , dc , and a variety of nonimmune cells and plays a pivotal role in the systemic inflammatory immune response by promoting the production of proinflammatory cytokines including tnf- and il-6 , which are involved in inflammatory and autoimmune diseases such as septic shock , cancer , inflammatory bowel disease [ 27 , 28 ] , rheumatoid arthritis [ 29 , 30 ] , obesity [ 31 , 32 ] , and diabetes [ 3335 ] .
moreover , mif has recently been proposed as a diagnostic biomarker for autoimmune diseases   such as arthritis [ 37 , 38 ] , type 2 diabetes , and ulcerative colitis   in both animals and humans .
the pathogenic contribution of mif to t1 dm was demonstrated by showing that mif mrna expression was upregulated in splenic lymphocytes during the development of spontaneously diabetic nonobese diabetic ( nod ) mice , as well as cyclophosphamide - treated nod mice .
diabetes incidence was increased to 86% in nod mice treated with recombinant mif ( rmif ) protein , compared with the 55% incidence observed in untreated control nod mice .
furthermore , studies performed using mif/ mice rendered diabetic by administering multiple low doses of streptozotocin ( stz ) have shown that the absence of mif affected several aspects of experimental t1 dm , including initial immunopathological events and the production of proinflammatory and cytotoxic mediators , thereby interfering with both inflammation and tissue destruction .
all the results described above provide evidence that mif plays a critical role in the pathogenesis of t1 dm .
however , the precise mechanism by which mif promotes insulitis and the mechanism underlying its proinflammatory role remain unclear .
the activities of mif may reside at the levels of both the inductive and effector phases of the inflammatory response attributed to antigen - presenting cells . here , we analyzed the influence of mif on m and dc activation using an autoimmune diabetes model in which multiple low doses of stz were administered to mif/ and wild - type ( wt ) mice ( mif/stz and wtstz , resp . ) in the balb / c genetic background .
as previously demonstrated mif/stz developed less severe hyperglycemia , reduced levels of ifn- and tnf- , a smaller amount of pancreatic islet antigen- ( piag- ) specific igg , and decreased cell infiltration into the pancreatic islets compared to wtstz .
interestingly , we found for the first time that m and dc from mif/stz displayed decreased expression of costimulatory molecules cd80 , cd86 , and cd40 , as well as toll - like receptor- ( tlr- ) 2 , tlr-4 , and major histocompatibility complex- ( mhc- ) ii .
importantly , we demonstrated that due to diminished upregulation of costimulatory molecules , these cells exhibited a reduced capacity to induce proliferation and cytokine expression in cocultures with allogeneic ovalbumin- ( ova- ) specific t cells .
all experiments in this study were performed according to the guidelines in the mexican regulations on animal care ( nom-062-zoo-1999 , 2001 ) and were approved by the local institutional animal care and use committee .
six- to 8-week - old male balb / c mice were purchased from harlan laboratories ( mexico city , mex . ) and were maintained as a breeding colony in a pathogen - free environment at our animal facilities in accordance with institutional guidelines .
abhay r. satoskar ( the ohio state university , usa ) and were maintained as breeding colonies for more than 10 generations in the balb / c genetic background on the transgenic mouse core facility at our institution .
genotyping of mif/ mice was routinely performed on dna isolated from tail snips using a pcr procedure .
the pcr were performed using the following primers : mif : forward 5-agaccacgtgcttagctgag-3 and reverse 5-gcatcgctaccggtggataa-3 ; neomycin ( neo ) : forward 5-attgaacaagatggattgcac-3 and reverse 5-cgtccagatcatcctgatc-3. pcr for the amplification of mif and neo was performed by adding 100  ng of the extracted dna to 25  l of a reaction mixture that contained 18.4  l of distilled water , 2.5  l of 10x pcr buffer , 0.4  l of dntps ( 10  mm ) , 1.5  l of mgcl2 ( 25  mm ) , 1  l of the forward and reverse primers , and 0.2  l ( 2.5 units ) of taq dna polymerase ( ampliqon , bioreagents and molecular diagnostics ) .
the amplification protocol consisted of 5  min at 95c , 35 cycles of 95c for 30  sec , 58c for 40  sec , and 72c for 30  sec and a final extension at 72c for 5  min .
a pcr fragment of 200  bp , corresponding to mif , or 500  bp , corresponding to neo , was visualized to identify wt or mif/ mice , respectively .
the pcr products were analyzed by electrophoresis on a 1.5% agarose gel and were viewed under uv light ( bio - rad , usa ) . 

mif/ and wt mice were deprived of food for 8  h before induction of diabetes via intraperitoneal ( i.p . )
injection of stz at doses of 40  mg / kg of body weight , daily for five consecutive days ( days 04 ) ( sigma - aldrich , st .
stz was diluted in cold 0.01  m citrate buffer ( ph 4.5 ) and was used within 5  min of preparation , in accordance with a previously reported protocol .
blood samples were collected by tail snipping from wt and mif/ mice that had been fasting for 6  h. samples were obtained once before stz injection and 2 , 4 , and 8 weeks after stz injection .
blood glucose levels were measured with a portable glucometer ( accu - chek sensor glucometer ; roche diagnostics , indianapolis , in , usa ) .
mice with a glucose concentration exceeding 300  mg / dl were considered to have t1 dm .
blood was collected and centrifuged at 1300  g , and the serum levels of mif ( neobiolab , usa ) , il-12 , ifn- , il-17 , il-4 , il-13 ( peprotech , mex ) , and insulin ( lincoln , st .
charles , mo , usa ) were determined via elisa according to the manufacturer 's instructions .
after isolation , the islets were lysed by five freeze - thaw cycles followed by sonication ( six 60-hz cycles for 1  min each ) on ice .
after centrifugation ( 10  000  g , 15  min , 4c ) , supernatants were collected and filtered through a 0.2  m membrane ( corning , cambridge , ma , usa ) .
the protein concentration was determined using the lowry method , and piag aliquots were stored at 70c until use .
serum samples were analyzed for the levels of pancreatic islet - specific th1-associated igg2a and th2-associated igg1 antibodies by elisa . briefly , 96-well elisa plates ( costar ) were coated with 100  l / well soluble piag in tris buffer , ph 7.8 .
, the wells were washed thoroughly with phosphate - buffered saline ( pbs ) containing 0.05% tween-20 ( pbs - tween ; merck , france ) and were blocked with pbs supplemented with 1% bovine serum albumin ( pbs - bsa ; sigma - aldrich ) for one hour at room temperature ( rt ) .
the serum samples were diluted 1  :  100 , followed by serial dilution of each sample ( in pbs - bsa ) from healthy or stz - treated wt or mif/ mice .
after extensive washing with pbs - tween , the samples were incubated for 45  min at rt with isotype - specific peroxidase - labeled goat anti - mouse antibodies ( anti - igg1 and anti - igg2a at 1/1000 dilutions ; zymed , san francisco , ca , usa ) . then
, the plates were washed , and immunoreactivity was detected with abts solution ( zymed ) .
pancreatic tissues from wt and mif/ mice healthy or injected with stz were removed , fixed overnight in 4% formaldehyde , and embedded in paraffin blocks .
afterwards , 5 to 7  m transverse sections of pancreatic tissue were sliced from the paraffinized tissue blocks , mounted on slides , and subsequently stained with eosin - hematoxylin ( e&h ; sigma - aldrich ) . for each mouse ,
one histological containing 13 nonsuccessive slices was scored for infiltration as previously described   according to the following scale : grade 0 ( no insulitis ) = 0% infiltration within the islets ; grade 1 ( peri - insulitis ) = 110% infiltration ; grade 2 ( moderate insulitis ) = 11<50% infiltration ; grade 3 ( severe insulitis ) = > 50% infiltration ; or grade 4 ( complete insulitis ) = extensive infiltration with few or no detectable pancreatic islet cells . using an olympus bx51 microscope ( olympus america , melville , ny , usa ) equipped with a digital video camera ,
spleen and pancreatic islet cells from wtstz and mif/stz were collected after 0 , 2 , 4 , and 8 weeks of injection with stz and stained for flow cytometry analysis .
briefly , the spleen was removed under sterile conditions , and spleen cells were obtained by mincing and filtering the tissue , followed by washing and suspension in dmem culture medium supplemented with 10% fetal bovine serum , 100 units of penicillin / streptomycin , 2  mm glutamine , and 1% nonessential amino acids ( all from gibco , brl , grand island , ny , usa ) .
spleen cells were suspended at 5  10  cells / ml in the same medium .
the pancreas was also removed under sterile conditions , and pancreatic islets were isolated using the collagenase method as previously described .
briefly , the pancreas was removed and cut into small pieces ( approximately 3  mm in size ) . the tissue was subsequently incubated in collagenase ( 0.3  mg / ml ; roche diagnostics corp . , indianapolis , in , usa ) for 10 minutes at 37c in a total of 1  ml of digestion solution under constant shaking and intermittent vortexing .
islets were subsequently washed several times in hbss containing bsa ( 5  mg / ml ) and were hand - picked under a dissecting microscope .
islets were dispersed into single cells by suspension in trypsin - edta ( gibco , brl ) and passage through a siliconized pasteur pipette .
cells from the spleen or pancreas obtained as described above were then used for flow cytometry analysis . in brief , cells were washed in flow cytometry wash solution ( dulbecco 's pbs containing 1% fcs and 0.05% sodium azide ) , followed by incubation with allophycocyanin- ( apc- ) conjugated anti - f4/80 and anti - cd11c antibodies for differentiation of m and dc , respectively . then
, the selected cells were incubated in 3% bsa - pbs containing a phycoerythrin- ( pe- ) labeled anti - cd80 , anti - ccr5 , or anti - tlr-4 antibody or a fluorescein isothiocyanate- ( fitc- ) labeled anti - cd86 , anti - cd40 , anti - mhc - ii , or anti - tlr-2 antibody ( all antibodies from biolegend , san diego , ca , usa ) at 4c for 30  min .
after incubation , the cells were washed several times in buffer , fixed in 1% paraformaldehyde ( sigma - aldrich ) , and stored at 4c in the dark , followed by analysis using a facscalibur flow cytometer and cellquest software ( becton dickinson , franklin lakes , nj , usa ) .
adherent m among peritoneal exudate cells ( pecs ) from healthy or 8 weeks after stz - treatment wt or mif/ mice were obtained .
briefly , the m density was adjusted to 5  10  cells / ml , and the cells were plated ( 100  l ) in 96-well flat - bottom plates ( costar , cambridge , ma , usa ) .
three hours later , the pecs were washed three times with warm sterile pbs to remove nonadherent cells , and 10  g of ova ( worthington , usa ) in 100  l of dmem supplemented media was added .
three hours later , adherent m were washed three times with warm sterile dmem to remove excess ova that had not been phagocytosed .
spleen cells from ova - transgenic mice were obtained as previously described , suspended at 1  10  cells / ml , and added ( 100  l ) to pecs at a ratio of 1 m  :  5 spleen cells .
then , [ 3h]-thymidine ( 185  gbb / mmol activity , amersham , uk ) was added at 0.5  ci / well , and the cells were incubated for a further 18  h. the cells were harvested using a 96-well harvester ( tomtec , toku , finland ) and then counted using a microplate counter ( trilux , toku , finland ) .
the values are presented as counts per minute ( cpm ) from triplicate wells . to establish the mif levels under conditions of t1 dm ,
the serum mif levels were determined weekly until 8 weeks after stz treatment in wtstz .
mif/stz received similar concentrations of rmif ( r&d systems , usa ) to the mif levels observed in wtstz to emulate physiological conditions in wtstz .
injection of stz together with 500  pg of rmif in 100  l of saline solution as a vehicle .
injection of rmif every three days at the following doses : 850  pg on week 1 ; 1280  pg on week 2 ; 1292  pg on week 3 ; 1305  pg on week 4 ; 4300  pg on week 5 ; 7300  pg on week 6 ; and , finally , 13200  pg on week 7 .
other mif/stz mice were injected with an equivalent volume of saline solution ( 100  l ) as controls .
comparisons between wt and mif/ mice that were healthy or treated with stz were performed using either student 's unpaired t - test or anova followed by turkey 's multiple comparisons test for data that displayed a normal distribution .
p values less than 0.05 were considered significant and were designated as p < 0.05 , p < 0.01 , or p < 0.001 .
all data were analyzed using graphpad prism 6 software ( san diego , ca , usa ) .
these mice sustained high blood glucose levels from week 2 ( 281.7  16  mg / dl ) until week 8 of stz administration ( 470.5  24  mg / dl ) ( figure 1(a ) , wtstz : squares ) .
in contrast , hyperglycemia developed gradually in mif/stz . the blood glucose level of mif/stz peaked at 219  23  mg / dl on week 4 , and the blood glucose level in some mice decreased between weeks 6 and 8 ( 226  15 and 182  16  mg / dl , resp . )
( figure 1(a ) , mif/stz : inverted triangles ) . this observation suggested that a slight recovery of the glucose response may occur in mif/stz at approximately week 8 of stz treatment .
wtstz showed a peak blood insulin level at 2 weeks after stz treatment , and after the sixth and eighth weeks of treatment , their blood insulin levels were significantly lower than those of healthy wt mice ( figure 1(b ) , wtstz : squares with solid line ; wt mice : white circles with dotted line ) .
interestingly , mif/stz showed no significant changes in insulin levels compared to healthy mif/ or wt mice over the course of the experiment ( figure 1(b ) , mif/stz : inverted triangles with solid line ; healthy mif/ mice : white triangles with dotted line ) .
we measured the serum levels of proinflammatory and anti - inflammatory cytokines in wt and mif/ mice after stz administration . as expected
, the wtstz displayed gradually increasing serum levels of inflammatory cytokines such as mif , il-12 , and ifn- ( figures 2(a ) , 2(b ) , and 2(c ) , resp .
the level of the proinflammatory cytokine il-17 was significantly increased at 8 weeks after t1 dm induction in wtstz ( figure 2(d ) , squares ) .
in contrast , mif/stz displayed significantly lower serum levels of mif , il-12 , ifn- , and il-17 than wtstz ( figures 2(a ) , 2(b ) , 2(c ) , and 2(d ) , resp . ;
the serum levels of the anti - inflammatory cytokine il-4 were significantly lower in wtstz ( squares ) than in mif/stz ( inverted triangles ) ( figure 2(e ) ) .
no differences in the serum il-13 levels were found between wtstz and mif/stz ( figure 2(f ) ) . to confirm that stz reached its target , wt and mif/ mice were treated with a single high dose of stz ( 150  mg / kg ) .
the toxic effect of high - dose stz was similar between wt and mif/ mice ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7053963 ) . in both experimental groups ,
the toxic effect of stz was present , and the high dose of stz destroys the insulin - producers beta cells , leading to acute nonimmune - mediated diabetes ; in contrast the multiple low doses of stz at 40  mg / kg , daily for five consecutive days , require participation of immune - inflammatory events for t1 dm development . to assess the damage to  cells in a model of t1 dm induced by multiple low doses of stz
, we sacrificed the mice at 8 weeks after t1 dm induction and removed the pancreas for h&e staining and histological analysis .
we assessed the number and size of normal and infiltrated pancreatic islets in each slide for the experimental and healthy mice .
islets from healthy wt and mif/ mice had a round morphology and well - defined borders without cellular infiltration ( figures 3(a ) and 3(c ) ) .
as expected , the pancreas from wtstz showed fewer and smaller islets than those from healthy wt mice . additionally , evident cellular infiltration led to the breakdown of islet morphology in the pancreas of wtstz ( figure 3(b ) ) .
in contrast , partial islet damage ( figure 3(d ) ) and no significant reduction in islet number or size were observed in mif/stz compared to wtstz ( table 1 ) . as shown in figure 3(e ) , mif/ mice at 8 weeks after stz administration displayed marked reductions in both invasive insulitis ( insulitis grades 3 and 4 ) and mild peri - insulitis ( insulitis grade 2 ) in pancreatic islets compared to wtstz .
thus , pancreatic islets from mif/stz had reduced damage associated with reduced leucocyte infiltration compared to pancreatic islets from wtstz .
these results demonstrated that stz administration triggers -cell destruction followed by insulin deficiency and hyperglycemia in wt mice .
although stz reached the pancreatic islets in mif/ mice , mif/stz exhibited minor damage compared to wtstz . in t1
dm , the immune system targets self - antigens within pancreatic islets and destroys the inhabiting insulin - secreting -cells .
therefore , autoantibody detection serves as a predictive factor for the onset of diabetes in both humans and mice [ 46 , 47 ] . to investigate how mif contributes to autoantibody production in t1 dm
, the serum levels of pancreatic islet - specific igg2a and igg1 antibodies were determined in wt and mif/ mice after 2 , 4 , and 8 weeks after stz administration .
wtstz produced high levels of igg2a at all time points analyzed ( figure 4(a ) ) and igg1 only at week 8 ( figure 4(b ) ) .
importantly , mif/stz mice displayed significantly lower levels of both igg2a and igg1 than wtstz ( figures 4(a ) and 4(b ) , resp . ) , suggesting suppressed development of an adaptive immune response .
these findings suggest that mif modulates the incidence and severity of diabetes by favoring the development of autoantibodies in t1 dm .
we showed that the reduced severity of t1 dm in mif/stz is associated with decreased pancreatic islet damage and diminished adaptive immune responses . to determine whether this phenotype could be related to the degree of activation of antigen - presenting cells
, we characterized the expression of the costimulatory molecules cd80 , cd86 , and mhc - ii and of the receptors tlr-2 and tlr-4 in m and dc from the spleen and pancreas of mif/ and wt mice after 0 , 2 , 4 , and 8 weeks of stz administration . in cells isolated from the spleen , the percentages of cd80- , cd86- , mhc - ii- , tlr2- , and tlr4-expressing m ( f4/80 ) were similar between healthy mif/ and wt mice ( figures 5(a ) , 5(b ) , 5(c ) , 5(d ) , and 5(e ) , resp . , at time 0 post - stz administration , right panel ) .
interestingly , upon stz administration , mif/ mouse m showed impaired activation , characterized by reduced expression of cd80 , cd86 , mhc - ii , tlr-2 , and tlr-4 , compared with m from wtstz mice ( figures 5(a ) , 5(b ) , 5(c ) , 5(d ) , and 5(e ) , resp . ) .
similarly , dcs ( cd11c ) isolated from the spleen of mif/stz mice expressed lower levels of cd80 , cd86 , and mhc - ii at 4 through 8 weeks after stz administration ( figures 6(a ) , 6(b ) , and 6(c ) , resp . ) , whereas the expression of tlr-2 and tlr-4 in dcs was reduced at 4 weeks in mif/stz compared to wtstz ( figures 6(d ) and 6(e ) , resp . ) . to investigate the role of mif in the activation of apcs in the pancreas , we determined mhc - ii and costimulatory molecule expression in m and dc in the pancreas of mif/stz and wtstz mice .
m from mif/stz expressed lower levels of cd80 and cd86 from 4 through 8 weeks after stz administration ( figures 7(a ) and 7(b ) , resp . ) .
alternatively , reduced expression of mhc - ii , tlr-2 , and tlr-4 was detected earlier , from 2 weeks until 8 weeks after stz treatment ( figures 7(c ) , 7(d ) , and 7(e ) , resp . ) .
moreover , dc from mif/stz mice displayed reduced cd80 at 4 to 8 weeks ( figure 8(a ) ) , cd86 at all time points analyzed ( figure 8(b ) ) , and reduced tlr-2 and tlr-4 expression at 4 weeks and 2 weeks , respectively , compared to dcs from wtstz ( figures 8(d ) and 8(e ) , resp . ) .
no significant differences in mhc - ii expression were detected ( figure 8(c ) ) .
the ability of m to activate t cells was investigated using ova - transgenic t cell cocultures .
m were collected from wt or mif/ mice treated or untreated for 8 weeks with stz . as shown in figure 9(a ) , after priming the cells with ova in vitro , m from mif/stz mice induced less t cell proliferation in response to ova than m from wtstz .
a similar trend was observed in cocultures of cd11b cells from mif/stz and ova - transgenic t cells ( figure 9(b ) ) .
these data suggest a role of mif in promoting m activation , which in turn induces specific t cell proliferation , particularly in this experimental t1 dm model .
the systemic levels of mif were reconstituted in mif/stz mice during the course of t1 dm , as described in section 2 . mif/stz that received rmif ( mif/stz+rmif ) displayed blood glucose levels similar to those in wtstz during the first six weeks after stz treatment . however , at 8 weeks after stz treatment , the glucose levels were not increased in mif/stz+rmif compared to wtstz ( figure 10(a ) ) .
comparable serum levels of the cytokines il-6 and il-12 were observed between mif/stz+rmif and wtstz during the first 6 weeks after stz treatment .
however , the levels of these inflammatory cytokines significantly decreased in mif/stz+rmif mice at 8 weeks after stz treatment compared to wtstz ( figures 10(b ) and 10(d ) ) .
interestingly , the serum levels of tnf- from mif/stz+rmif were higher than those of wtstz at all time points analyzed ( figure 10(c ) ) .
these results confirm that mif acts as a powerful inducer of proinflammatory cytokines involved in the development of experimental t1 dm .
currently , there is no doubt that mif is a key molecule that promotes proinflammatory immune responses .
this proinflammatory property of mif contributes to developing protective inflammatory - th1 immune response in different models of parasitic diseases .
in contrast the same proinflammatory property of mif participates in the pathogenesis of many inflammatory diseases . in line with the last one , recently it has been established that high blood levels of mif are associated with human t1 dm , similar to the findings in experimental mouse models of t1 dm [ 20 , 21 , 24 , 51 ] .
studies in nod mice or mice treated with multiple low doses of stz ( despite the pathogenic differences between these models ) have shown that pancreatic  cell destruction results from the toxic effect of free radicals ( o2 , h2o2 , and nitric oxide ) and inflammatory cytokines released by activated m and t cells [ 15 , 41 , 52 ] .
therefore , both models have been widely used to dissect the role of mif in the pathogenesis of t1 dm using anti - mif monoclonal antibody treatment or using mif/ mice . in all cases ,
the lack of mif resulted in diminished manifestation of the disease , decreased glucose blood levels , and reduced production inflammatory cytokines associated with the development of t1 dm , including tnf- , il-1 , ifn- , il-12 , and il-23 [ 21 , 22 , 24 ] .
our study validates and extends these findings by demonstrating an important role for mif in promoting costimulatory molecule expression in m and dc during t1 dm development .
we further demonstrate that , in addition to regulating m and dc activation in t1 dm , m isolated from t1 dm mif/ mice exhibit reduced t cell activation . here
, we observed that wt mice treated with stz exhibited high blood glucose levels greater than 400  mg / dl but mif/stz mice developed lower glucose levels of approximately 200  mg / dl , in association with lower serum levels of proinflammatory cytokines .
after reconstituting mif using exogenous rmif , mif/stz showed blood glucose levels and il-6 and il-12 levels similar to those in wtstz from 2 to 6 weeks after stz treatment .
interestingly , tnf- serum levels from mif/stz+rmif were higher than those of wtstz at all time points analyzed .
the reduction levels of blood glucose , il-6 , and il-12 observed in mif/stz mice , on week 8 , probably were because the animals did not receive rmif injections on week 8 , as mentioned in section 2 , so the residual effect of rmif from week 1 to week 7 was insufficient to induce il-6 and il-12 levels at 8 week similar to that observed in the wtstz mice in this point , at least for these two cytokines .
these observations confirm that mif exogenous acts as a powerful inducer of il-6 and il-12 , but only if it is present in steady high concentration .
in addition , we found that mif/stz did not produce detectable levels of islet autoantibodies , in contrast to wtstz , which produced high levels of islet autoantibodies .
it is well known that the early presence of islet autoantibodies is decisive in the development of diabetes by nod mice as well as humans [ 46 , 47 , 53 ] .
our results confirm that mif is essential for the development of hyperglycemia and suggest a role for mif not only in the innate immune response but also in the adaptive immune response in t1 dm .
recently , it has been reported that mif is produced by pancreatic -cells and that mif is released by insulin granules in an autocrine fashion [ 54 , 55 ] .
the chemical destruction of -pancreatic islets by stz in wt mice damaged -pancreatic islets ; this condition could reduce one major source of mif in this experimental t1 dm model .
however , we did not observe a reduction in mif levels in this model ; in contrast , wt mice produced high serum levels of mif after stz administration . in line with this finding , it is known that the pancreatic islets remaining after treatment with stz produce high levels of mif   and that an elevation of mif secretion precedes pancreatic islet death induced by ifn- , tnf- , and il-1 .
this evidence establishes that stz did not influence mif production / release by pancreatic islets or other cellular sources , such as t cells , dc , and m infiltrating the pancreas .
the loss of insulin production in t1 dm is related to pancreatic -cell destruction due to insulitis .
we observed that wtstz developed high serum levels of mif and low insulin levels compared to mif/stz , which expressed insulin levels comparable to those in healthy mice .
the histological analysis of pancreatic islets showed that wtstz displayed 100% insulitis , compared to the 33% insulitis observed in mif/stz mice .
these observations confirm that mif deficiency resulted in pancreatic islet protection , probably by controlling the functional activity and modulating the secretory capacity of proinflammatory cytokines produced by t cells , dc , and m that reach the pancreatic cells .
mif has been recognized as a molecule that not only promotes proinflammatory cytokine production but also acts as a chemokine .
for example , mif plays a crucial role in leukocyte recruitment and arrest during atherosclerosis development .
therefore , mif could participate in the process of insulitis to promote the production of proinflammatory cytokines , but mif could also promote leukocyte recruitment to pancreatic -cells .
antigen - presenting cells , m and dc , are key mediators of the development of t1 dm . moreover , it has been proposed that dc orchestrate the autoimmune response in t1 dm via tlr-2 and tlr-4 .
previous studies by us and others have shown that mif induces the expression of costimulatory molecules on m and dc in some pathological infections [ 5961 ] . here
, we identified the expression of costimulatory molecules and tlr-2 and tlr-4 on m and dc , as well as the ability of m to activate t lymphocytes .
our results demonstrated that both m and dc from the spleen and pancreas of mif/stz mice expressed lower levels of cd80 , cd86 , mhc - ii , tlr-2 , and tlr-4 than those of wtstz .
these results demonstrate a role of mif in the activation of m and dc to promote costimulatory molecule expression , which might drive pancreas - specific t cell activation and effector th1 subset differentiation , processes that have been associated with subsequent pancreatic injury in t1 dm . in line with the results described above
, we observed that both m ( f4/80 ) and monocytes ( cd11b ) from wtstz were more reactive and had greater ability to induce t lymphocyte - specific proliferation in response to ova than those from mif/stz mice .
this finding is consistent with the evidence that proliferation of antigen - specific t cells from tcr - transgenic mice is highly dependent on cd28/cd86 costimulation .
this conclusion agrees with the result that blocking cd86 prevents the development of diabetes in nod mice .
moreover , mutations in the mhc - ii molecule lead to development of autoimmune diabetes . by another hand
, the differentiation state of m is an important determinant for t cell response in t1 dm .
two major populations have been defined , the classically activated ( ca ) m secreting proinflammatory cytokines such as tnf- , il-6 , and il-1 and reactive oxygen species ; and alternatively activated ( aa ) m which secrete anti - inflammatory factors including tgf- and il-10 . in t1 dm
it has been established that cam trigger inflammatory responses which initiates insulitis and pancreatic  cell death , whereas the aam decreases hyperglycemia , insulitis , and inflammation in the pancreas .
as mif is a regulator of many proinflammatory cytokines that are characteristic for the cam , mif has been proposed as cam macrophage - polarizing factor .
however , there are few and contrary experimental evidences about how mif might participate in the polarization to aam or cam. in a mouse model of nonalcoholic fatty liver disease m in liver from mif/ mice were skewed toward aam . by contrary , in the melanoma mouse model m-derived mif participates in aam polarization .
moreover , murine mif and filarial nematode parasite ( brugia ) mif protein induced proinflammatory cytokines release .
however , mif also induce upregulation of il-4 on bone marrow - derived mouse m , which when treated in vitro with mif and il-4 induce aam . here , we show that m from mif/stz mice display reduced proinflammatory cytokine production and exhibit reduced ability to induce t lymphocyte proliferation in response to ova .
it is possible that mif deficiency influences on aam polarization in this model ; however , more experiments are necessary to establish this .
we show for the first time a role of mif in promoting costimulatory molecule expression in m and dc in t1 dm .
these results reveal mif as a key regulator of proinflammatory function and m and dc activation in t1 dm .
although more specific experiments are required , there is no doubt that mif represents a potential target for anti - mif therapy , which might attenuate the autoimmune process in t1 dm .
 macrophage migration inhibitory factor ( mif ) is highly expressed in type 1 diabetes mellitus ( t1 dm ) . 
 however , there is limited information about how mif influences the activation of macrophages ( m ) and dendritic cells ( dc ) in t1 dm . to address this issue , we induced t1 dm by administering multiple low doses of streptozotocin ( stz ) to mif/ or wild - type ( wt ) balb / c mice . 
 we found that mif/ mice treated with stz ( mif/stz ) developed lower levels of hyperglycemia , inflammatory cytokines , and specific pancreatic islet antigen- ( piag- ) igg and displayed reduced cellular infiltration into the pancreatic islets compared to wt mice treated with stz ( wtstz ) . 
 moreover , m and dc from mif/stz displayed lower expression of mhc - ii , costimulatory molecules cd80 , cd86 , and cd40 , toll - like receptor- ( tlr- ) 2 , and tlr-4 than wtstz . 
 these changes were associated with a reduced capacity of m and dc from mif/stz to induce proliferation in ovalbumin - specific t cells . 
 all the deficiencies observed in mif/stz were recovered by exogenous administration of recombinant mif . 
 these findings suggest that mif plays a role in the molecular mechanisms of m and dc activation and drives t cell responses involved in the pathology of t1 dm . 
 therefore , mif is a potential therapeutic target to reduce the pathology of t1 dm . 