unilocular adipocytes are the 
     principal component of white adipose tissue and have a classical role in the regulation of 
     triglycerides and fatty acid accumulation during energy expenditure and deprivation 1 - 2 .
the regulation of 
     adipocyte differentiation is controlled by several factors , including hormones and their 
     receptors that activate proteins and transcription factors such as cebp/ , 
     ppar- , cebp/ , ap2 and others 3 - 4 . besides unilocular adipocytes , mesenchymal stem cells and 
     differentiating adipocytes
have been described in the adipose tissue making it appear that 
     mesenchymal stem cells under specific stimuli become committed to the adipocyte lineage and able 
     to accumulating lipids thus forming adipocytic multilocular cells , preadipocytes , and 
     ultimately , unilocular adipocytes 1 - 4 .
more recently , several groups have demonstrated that adipose tissue is able to produce 
     proinflammatory cytokines and fat - derived peptides termed adipokines ( including ligands such as 
     adiponectin , leptin , resistin , tnf- , il-6 ) , which act in a paracrine , autocrine and/or 
     endocrine manner 4 - 5 .
the accumulation of adipocytic cells and an increased percentage of fat in several 
     ectopic regions of the body with aging have been well - described 6 .
this increase also appears to correlate with observed increases in circulating levels 
     of proinflammatory cytokines during aging 7 .
this is of 
     great interest from an immunological perspective , as the thymus is one of the organs known to 
     accumulate fat during aging and has been shown to be sensitive to inflammatory changes 8 - 10 .
the thymus is a primary lymphoid organ responsible for the differentiation and maturation of t 
     lymphocytes 10 - 12 . 
    
anatomically , it is a bi - lobed organ subdivided in lobules by septa that emerge from the 
     capsule .
blood vessels and nerves are able to reach the thymic parenchyma by the septa region . 
     in the young thymus ,
each lobule contains two very well defined regions : the cortex , enriched in 
     immature t cells ; and the medulla , where mainly mature immunocompetent thymocytes can be found , 
     before exiting the organ to populate the periphery .
the process of t cell maturation initiates 
     during fetal development . in post - natal life , progenitors that originated in the bone marrow 
    
enter into the thymus and interact with several different thymic stromal cell types , including 
     epithelial cells , macrophages , dendritic cells and fibroblasts , which participate of the t cell 
     differentiation process 10 - 12 . with age ,
the thymus gradually decreases its capacity to generate 
     immunocompetent t cells and becomes minimally functional , as it undergoes dramatic changes in 
     its size , morphology and cell composition , a process termed  age - associated thymic 
     involution  8 - 10 , 13 - 16 . 
    
decreased thymopoiesis , loss of cortical and medullary boundaries , deposition of unilocular 
     adipocytes and changes in the expression of various thymic factors have been shown to occur 
     during this process .
this is associated with the increased susceptibility of aged individuals to 
     infectious diseases 10 , 17 .
thus , investigating the mechanisms that regulate thymic involution and identifying 
     the cells that participate in this process might contribute to the development of strategic 
     therapies for immunodeficiency conditions , as in the case of aging .
in the current studies , we have investigated the distribution of fat - storing cells in the 
     aging thymus and we found not only an increase of unilocular adipocytes but also an increase of 
     adipocytic - like multilocular mesenchymal cells in the septa and parenchymal regions of the 
     organ .
these findings suggest that adipocyte precursors or fat - storing cells may be migrating 
     into and/or differentiating actively within the aging thymic microenvironment .
furthermore , we 
     found that the adipocyte - like cells accumulating in the thymus with age express the chemokine 
     receptor , ccr5 . using the well - characterized adipocytic mesenchymal cell line , 3t3-l1
, we found 
     that ccr5 ligands are capable of regulating the migration and differentiation of these cells and 
     suggest a potential role for these chemokines in adipocyte biology .
balb / c mice bred in the national institute on aging rodent colony ( bethesda , md ) were 
     utilized at 2 , 4 , 6 , 9 , 12 , 18 , 21 and 24 month - old .
mice were housed in environmentally 
     controlled rooms with a 12h light - dark cycle according to the procedures outlined in the 
     " guide for the care and use of laboratory animals " [ nih publication no . 86 - 23 , 
     1985 ] .
ccr5-deficient mice ( b6;129p2-ccr5/j ) originally 
     obtained from jackson laboratories ( bar harbor , me ) were aged in the animal house of the niaid 
     and kindly donated by dr .
1,152 cdna clones were selected from a verified sequence master set containing 15,000 
     human t1 phage - negative image consortium clones commercially obtained from research genetics , 
     inc .
nylon membranes were used as substrate for denatured cdna clone printing 
     in duplicates , using a gms417 microarrayer ( affymetrix , santa clara , ca ) . before the addition of 
     the cdna probe , the thymi of 2- , 4- , 6- , 12- and 18-month - old balb
/ c mice were placed into 
     multiple pools by age for total rna extraction using rnazol b ( tel - test , friendswood , tx ) and 
     cdna probes were prepared using reverse transcriptase .
cdna microarray membranes were loaded 
     into conical tubes containing mycrohyb hybridization buffer ( research genetics ) in presence of 
     0.6g/l of cot1 dna ( life technologies ) and 0.5g/l of 
     poly a primers ( sigma ) blocking agents .
after prehybridization , p - labeled cdna probes ( at the concentration of 
      1x10 counts / ml hybridization buffer ) were added to the tubes with cdna microarray 
     membranes and hybridized overnight at 42c .
hybridized membranes were washed 
     twice with 15 ml of wash solution ( 0.1%sds and 2x ssc ) for 15 min at 55c 
     and at room temperature , respectively .
the radioactive cdna microarray membranes were examined 
     on a phosphorimager ( molecular dynamics storm , sunnyvale , ca ) at a resolution of 50 m . 
    
grid overlays were utilized to identify cdna targets on the arrays and signal intensity for each 
     cdna was examined using the array pro software ( media cybernetics , silver spring , md ) . 
    
background correction for each cdna microarray hybridization assay was assessed via the 
     subtraction of single spot intensities by the median of the background signal intensity from the 
     array .
the background - subtracted spot intensities were subsequently transformed to loge scale and ratio comparisons were done using the group of 2 
     month - old as control .
1g of total rna isolated from the thymi of 2- , 
     12- or 18-month - old balb / c mice was utilized to generate cdna probes using taqman reverse 
     transcription reagents ( pe applied biosystems , foster city , ca ) .
the sybr green i assay and the 
     geneamp 5700 sequence detection system ( pe applied biosystems ) were utilized for the detection 
     of real - time pcr products as previously described 18 . 
    
primers were designed for ccr5 , ccl3 , ccl4 and ccl5 based on their sequence in genbank 
      ( www.ncbi.nlm.nih.gov/genbank ) as 
     well as for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , which was utilized as control . for 
     each of the age groups ,
pcr reactions were performed in duplicate in a 96-well plate for each 
     gene - specific primer pair tested .
the comparative threshold cycle 
     ( ct ) method ( pe applied biosystems ) was utilized to determine 
     relative quantity of gene expression for each gene compared with the gapdh control .
briefly , 
      ct values from gapdh reactions were averaged for each duplicate and 
     the relative difference between gapdh and each duplicate was calculated ( 2 
      ct gapdh - ct experimental ) .
this value 
     was then averaged for each duplicate set and divided by the value of the 2 month - old thymus 
     samples to determine the relative fold induction for each sample .
differences regarding fold 
     change values observed between the cdna microarray and real - time pcr results might be due to 
     experimental variability in the two distinct assays .
thymi ( n=4 ) of 2 , 9 , 12 , 18 and 21 months of age were mounted in a tissue array as 
     previously described 19 . for hematoxylin and eosin 
     staining
, sections were deparaffinized in xylene , re - hydrated in graded alcohols and placed at 
     high temperature in solution of 0.01 m sodium citrate buffer ( ph 6.0 ) for 40 min .
sections were 
     then stained with hematoxylin ( lerner laboratories ) , rinsed and differentiated with 1% acidic 
     alcohol before eosin ( lerner laboratories , pittsburgh , pa ) staining , dehydrated in graded 
     alcohol and then mounted in organic media . for peroxidase immunohistochemistry ,
a standard 
     two - step dako autostainer ( dako corporation , carpenteria , ca ) was utilized to examine the thymic 
     sections .
primary purified rabbit anti - mouse ccr5 ( bd pharmingen , ca ) , rabbit anti - ccl3 , -ccl4 
     ( r&d systems , mn ) or -ccl5 ( chemicon , ca ) igg antibodies were applied on thymic sections 
     at 10g / ml for 60 min at room temperature in moist humidified chamber .
after extensive 
     washing , tissue sections were incubated with peroxidase - conjugated donkey anti - goat , goat 
     anti - rabbit or rabbit anti - rat ( santa cruz biotechnologies ) antibody , for 30 min , after which 
     the sections were extensively washed and then immersed in a freshly prepared chromogen / substrate 
     reagent ( diaminobenzidine , dab/ hydrogen peroxide , h2o2 ) .
slides were 
     mounted using an organic media ( cytoseal60 , stephens scientific , riverdale , nj ) . 
    
pbs , ph 7.4 , was used for all intermediate wash steps . for immunofluorescence , rat anti - mouse fibroblast/
mesenchymal cells ( er - tr7 ) ( novus 
     biologicals , littleton , co ) igg antibody was used at 10g / ml for 60 min .
after pbs 
     washing , slides were incubated with anti - rat igg antibody conjugated to alexa-594 ( molecular 
     probes , eugene , or ) .
subsequently , slides were washed , incubated with the dna dye dapi 
     ( 4',6-diamidino-2-phenylindole ) and mounted using glycerol 50% aqueous mounting media .
oil red o ( fischer scientific , hanover park , il ) was diluted in 50% propylene glycol , and 
     filtered .
thymic tissue sections were incubated with oil red o solution for 4h at room 
     temperature and then rinsed with 50% isopropyl alcohol and in distilled water before hematoxylin 
     staining , for 2 min .
slides were then rinsed with ammonia 5% in water , blot dried and mounted 
     with glycerol 50% aqueous mounting media .
cells were cultured in dulbecco 's minimal essential medium ( dmem ) supplemented with 10% 
     calf serum until confluence ( 6 - 7 days ) .
then , confluent 3t3-l1 preadipocytes were treated for 9 
     or 12 days with 100 ng of ccl3 , ccl4 or ccl5 ( chemicon , ca ) .
subsequently , differentiation of 3t3-l1 cells was analyzed by lipid accumulation 
     under light microscope and by western blot analysis for adipocyte differentiation markers .
3t3-l1 cells were lysed in 1.5x laemlli sample buffer and proteins were quantitated by 
     bradford reagent ( bio - rad , hercules , ca ) . after heating for 5 min at 95c , proteins 
     were separated by sds - page on 12% polyacrylamide gel and transferred onto 0.22m 
     polyvinylidine ( difluoride ) membranes ( invitrogen ) .
membranes were probed with rabbit 
     anti - ppar2 ( affinity bioreagents , co ) or rabbit - anti - ap2 ( genetex , tx ) antibodies . 
    
signal detection was performed by chemiluminescence ( ecl ) using hyperfilm ( amersham 
     biosciences ) .
3t3-l1 preadipocytes were placed in fibronectin - coated plates ( bd biosciences , ca ) in 
     dmem medium .
after two days post - confluence , cells were treated in the absence or in the 
     presence of ccl3 , ccl4 or ccl5 ( 100 ng / ml ) for 16h . a wound was inflicted with a sterile plastic 
     tip by scratching the confluent monolayer .
cells moving into the scratch were observed over 
     several time points using light microscopy .
tissue arrays were used to characterize the phenotype of adipocytic cells within the thymus of 
     aging mice .
histological and morphological analyses indicated an age - dependent increase in 
     adipose tissue deposition , including unilocular adipocytes and multilocular cells .
upper panels 
     of figure 1 represent the thymus of 2 , 12 and 21 month - old 
     mice stained for hematoxylin and eosin . in the thymus of 2 month - old mice the cortical and 
     medullary regions
were well - defined and very few or no adipocytes were observed in the septa 
     region or inside the organ ( figure 1a ) .
a progressive 
     deposition of unilocular adipocytes was mainly seen in the thymic perivascular space ( pvs ) , as 
     shown in the representative photomicrography of 12 month - old murine thymus ( figure 1b ) .
interestingly , in some thymic tissue sections of 18 
     month - old animals , isolated adipocytes were observed in the subcapsulary region of the organ 
     ( not shown ) and fibroblastoid cells were visualized inside the thymic parenchyma .
these 
     fibroblastoid cells were observed connected to the capsule , thus creating a boundary - like 
     structure in the subcapsulary region of the organ ( figure 3h ) . as expected , as thymus aged , a decrease in the number of thymocytes
was seen in the 
     cortex and loss of cortical and medullary boundaries were observed in the thymic lobules ( figure 
      1 ) . to further investigate the presence of fat - storing cells in the aging thymus , we performed oil 
     red o staining in frozen mouse thymic tissues . increased number of multilocular oil red 
     o cells
while the thymi of 2 month - old mice showed few , if any , oil red 
     o multilocular cells , mice at 4 and 6 months of age presented fat - storing cells 
     mainly in the septa region of the organ ( not shown ) .
moreover , oil red o+ 
     multilocular cells were observed in the thymic septa and in the thymic capsular regions of 
     12 month - old mice .
finally , a higher number of oil red o multilocular cells were 
     visualized in the septa region and inside the thymic parenchyma of 12 and 24 month - old thymi 
     ( figure 1 ) .
these data lead us to propose that 
     adipocytic / mesenchymal cells may be migrating to the aging thymus and behaving as a thymic 
     stromal cell component able to interact closely with differentiating thymocytes and other 
     microenvironmental cells in the aging thymus .
age - dependent thymic expression of ccr5 chemokine receptor . based on the 
     possibility that adipocytic / mesenchymal cells could be immigrating into the thymus
, we looked 
     for genes that significantly changed with age and are known to be involved in cell migration . 
    
using cdna microarray technology , changes in thymic gene expression profiles were analyzed as a 
     feature of aging .
one of the genes identified was ccr5 , a chemokine receptor known to bind to 
     the proinflammatory chemokines ccl3 , ccl4 and ccl5 .
previous data have demonstrated that ccr5 
     participates in t cell migration and activation 20 . in 
     addition
, ccr5 has been shown to be expressed in t cells , macrophages and , interestingly , human 
     adipocytes and 3t3l1 preadipocyte cell line 21 - 23 .
we analyzed the expression of ccr5 mrna and found that it 
     was increased in the aging thymus , as detected by cdna microarray and confirmed by real time 
     rt / pcr ( figure 2 ) .
flow cytometric analyses revealed that 
     only 1 to 2% of total mouse thymocytes express ccr5 , independently of age ( data not shown ) , 
     indicating that this increase may be attributable to ccr5 production by thymic stromal cells .
ccr5 expression in the aging thymus is mainly detected in fat - storing multilocular 
      cells . to substantiate the above observations
, we performed immunohistochemical analysis 
     of our thymic tissue array and demonstrated an increase of ccr5 expression in the aging thymus 
     ( figure 3 ) . although some thymocytes stained positive for 
     ccr5
were observed , the main thymic cell type expressing ccr5 resembled adipocytic - like 
     multilocular cells or fat - storing cells .
these cells were mainly found in the septa region , 
     adjacent to adipocytes , and also in the thymic parenchyma , interacting with thymocytes and 
     thymic microenvironmental cells ( figure 3 ) . 
    
morphologically , ccr5 multilocular cells and fibroblastoid cells were present in the 
     perithymic adipose tissue as well as in the perivascular space in the septa and capsule . 
    
additionally , these cells were visualized in the subcapsulary region of aged thymi and inside 
     the thymic aged parenchyma in contact with thymocytes ( figures 3 ) .
serial sections of thymic tissue arrays showed that these multilocular cells stained 
     positively for er - tr7 , a known marker for murine mesenchymal cells ( figure 4 ) .
to further 
     investigate a possible role for ccr5 on immigration of fat - storing cells , we examined the 
     expression of ccr5 ligands in the aging thymus .
real time - rt - pcr analysis showed that the 
     expression of ccl3 , ccl4 and ccl5 mrna increased in thymus and total thymocytes with age ( figure 
      5 ) . using immunohistochemical method ,
thymocytes and 
     thymic stromal cells stained positively for ccl4 and ccl5 ( figure 6 ) .
interestingly , in the septa region of the thymus of 2 month - old mice , ccl4 
     expression was detected in cells resembling pericytes and myofibroblasts , mesenchymal cell types 
     ( figure 6 ) . in the thymus of 12 months of age , ccl4 
     expression was observed in thymocytes and in multilocular cells in the septa and inside the 
     thymic parenchyma .
these data reinforced the hypothesis that adipocytic mesenchymal cells could 
     be possibly immigrating and/or differentiating in the aging thymus by an active process 
     regulated by ccr5 ligands .
the 
     possibility that ccr5 ligands could be influencing immigration of adipocytic mesenchymal cells 
     into the aging thymus lead us to investigate whether oil red o cells could be 
     visualized in thymi of 10 - 11 month - old ccr5-deficient mice . both aged ccr5-deficient mice and 
     wild type control group presented oil red o multilocular cells in the septa and in 
     the cortical region of the thymus ( figure 7 ) . although the 
     number of oil red o cells in ccr5-deficient mice was lower than in control group , 
     this difference was not statistically significant . moreover , there was no significant 
     histological change between ccr5-deficient and control thymi stained for hematoxylin and eosin 
     ( data not shown ) .
ccr5 ligands regulate differentiation and migration of 3t3-l1 adipocytic multilocular 
      mesenchymal cells in vitro . due to the lack of specific surface markers 
     for adipocytic multilocular mesenchymal cells and technical difficulties to isolate them
, we 
     chose to analyze the effects of ccr5 ligands in murine 3t3-l1 cells , a well - characterized 
     embryonic mesenchymal cell line known to differentiate into adipocytes in vitro 
      24,25 . 
     to investigate the role of ccr5 ligands in 3t3-l1 cells , immunocytochemistry was used to verify 
     the expression of er - tr7 and ccr5 .
in addition , 3t3-l1 cells in 
     differentiation also expressed ccl3 , ccl4 and ccl5 , thus suggesting that these chemokines could 
     possibly act in an autocrine and paracrine manner ( figure 8a ) .
the differentiation of 3t3-l1 cells is routinely carried out by the addition of dexamethasone , 
     insulin and isobutyl - methyl - xanthine to the culture medium 24,25 . however , in the absence of these specific 
     factors , 3t3-l1 cells are able to differentiate spontaneously although to a much slower rate . to 
     analyze if ccr5 ligands would be able to stimulate differentiation of 3t3-l1 cells to 
     adipocytes , ccl3 , ccl4 or ccl5 was added to the culture medium of confluent cells for nine or 
     twelve days .
these chemokines were found to stimulate the differentiation of 3t3-l1 cells , as 
     evidenced by the increase expression of the adipocyte differentiation markers , ppar2 
     and ap2 ( figure 8b ) . by the ninth day of culture ,
the 
     number of refringent multilocular cells accumulating lipids was higher in ccr5 ligand - treated 
     cells when compared to untreated cells .
this observation was even more evident at twelve days of 
     culture ( figure 8c ) .
we also investigated the influence of ccl3 , ccl4 and ccl5 on the migration of multilocular 
     mesenchymal cells in vitro .
3t3-l1 cells were cultured in fibronectin 
     coated - plates and two days post - confluence , cells were treated with ccl3 , ccl4 or ccl5 for 20h , 
     followed by scratching of the plate . as indicated in figure 9a , after 20h
, higher cell motility was observed in the plates treated with all three 
     ccr5 ligands , as compared to untreated cells , and the treatment of ccl5 elicited the strongest 
     effect .
taken together , these results show that ccr5 ligands are able to modulate adipocyte 
     differentiation as well as the migration of 3t3-l1 adipocytes .
within the young thymus , several cell types have been described as playing a role in thymic 
     physiology including thymocytes , thymic epithelial cells , dendritic cells , fibroblasts , 
     mesenchymal cells and a variety of hematopoietic cells , such as macrophages , nk and b cells 
      25 , 26 .
however , 
     alterations in numbers , distribution and function of these thymic cellular components during the 
     aging process are poorly known . in this work
, we suggest that adipocytes and fat - storing cells 
     may be an active component during thymic atrophy associated with age . in this context , 
     unilocular adipocytic cells have been described to fill the space left by the receding lymphoid 
     thymic compartment as thymus age 9 , 27 , 28 .
it is known that adipocyte 
     deposition occurs in the aging thymus possibly due to the loss of thymopoiesis and increase of 
     thymic interlobular spaces , characteristics of thymic involution 10 , 27 , 28 . in agreement , our histological analysis show progressive age - dependent alterations 
     in the thymic organ , with unilocular adipocytes accumulation in the mouse thymic perivascular 
     space 9 , 27 - 29 .
in addition , our data also show interaction between 
     unilocular adipocytes with thymocytes and thymic stromal cells inside the thymic parenchyma . 
    
these observations suggest a possible functionality to adipocytic cells in the thymus which 
     conflicts with the general belief that during aging , adipocytic unilocular cells simply fill a 
     space left by the thymic lymphoid compartment or replace the lymphocytic perivascular space . in the present study , we defined not only thymic unilocular adipocytes in the thymus but also 
     fat - storing multilocular mesenchymal cells , based on their morphology , oil red o and ertr7 
     staining .
we found that the presence of fat - storing cells inside the murine thymic parenchyma 
     and septa region with age is in accordance with a previous study showing the ultrastructure of 
     adipocytic multilocular cells in the thymus of rats and mouse 30 .
interestingly , oil red o cells were shown within the perivascular space of human thymus 31 . however , these authors failed to comment on the presence of adipogenic 
     multilocular cells within the aged human thymus .
an increased number of fat - storing multilocular cells within the aging thymus were also 
     observed in our study .
however , how these cells appear in the thymus and their function is 
     unknown .
one 
     hypothesis is that adipogenic multilocular cells could be differentiating from mesenchymal cells 
     known to be present in the thymic parenchyma since fetal life 32 .
it is also possible that fat - storing multilocular cells could be differentiating 
     from pericytes associated with endothelial cells in blood capillaries of the thymus . in this 
     context
, pericytes have the potential to differentiate to distinct mesenchymal cell lineages , 
     including the adipocytic one 33 , 34 .
third , the immigration of adipocytic precursors from the perithymic 
     adipose tissue or circulation may result in adipocytic multilocular cell accumulation in the 
     aging thymus .
recently , bone marrow mesenchymal stem cells were found to migrate from the 
     circulation to several tissues , including the thymus 35 , 
      36 .
finally , the possibility that all these events are 
     happening conjointly during the process of aging can not be ruled out .
our results show an age - associated increase in ccr5 mrna expression in the thymus that 
     correlates with an increased number of ccr5 multilocular cells within the thymic 
     capsule , septa and in the parenchyma of the organ . in this context , ccr5 expression has been 
     previously found in human adipocytes in vitro and in human adipose tissue 
      in situ as well as in 3t3l1 preadipocyte line in culture 25 , 37 .
thus , the possibility that the 
     chemokine receptor ccr5 could be regulating adipocyte migration was investigated . toward this 
     end ,
the ccr5 ligands , ccl4 and 
     ccl5 , increase in the thymic parenchyma as revealed by the age - associated increases in the 
     staining of thymocytes and microenvironmental cells , including the multilocular fat - storing 
     cells .
these data suggest that chemokines may influence the migration and possibly the 
     differentiation of adipogenic or fat - storing cells within the aging thymus . however , our 
     observations showing no significant differences in the number of fat - storing multilocular cells 
     using 11 month old ccr5-deficient versus wild type mice ( although a trend of lower adipocyte 
     numbers was observed ) failed to support a role for ccr5 in adipocyte development in the aging 
     thymus . however , these data also raised two possibilities .
first , the progenitors of these 
     adipocyte - like cells could be thymic mesenchymal cells that are known to be present in the 
     thymus parenchyma 31 and whose phenotype may be modified 
     during the aging process .
second , other chemokine receptors could be acting in the migration of 
     adipocytic mesenchymal cells to the aging thymus of ccr5-deficient mice .
however , it should be 
     noted that we were quite limited with the number of aged ccr5 deficient mice available to us to 
     examine here and additional studies may provide more conclusive data regarding the role of 
     distinct chemokine receptors in adipogenic mesenchymal cell immigration and/or differentiation 
     within the aging thymus .
interestingly , we have found that the ccr5 ligands , ccl3 , ccl4 and ccl5 , without any other 
     stimuli , were able to induce adipogenesis using the 3t3-l1 mesenchymal cell line , as evaluated 
     by the increased expression of ppar2 and ap2 38 , 39 . to our knowledge , this is the first time 
     ccr5 ligands are described to stimulate adipocyte differentiation .
while these data do not 
     directly demonstrate the ability of chemokine ligands to facilitate primary mesenchymal cell 
     differentiation to the adipocyte lineage , these experiments support the idea that chemokines may 
     be possibly acting as chemoattractants for adipogenic mesenchymal and/or fat - storing cells , 
     facilitating their immigration and possibly their subsequent differentiation within the aging 
     thymic microenvironment .
although additional work will be needed to establish a role for 
     adipogenic cells in the thymus , several groups have shown thymic atrophy in transgenic mice 
     harboring defects in adipocyte physiology , such as ob / ob ( leptin ) , db / db ( leptin receptor ) and 
     corticotropin - releasing factor overexpressing mice 40 , 
      41 . in this context , reducing proadipogenic signaling in 
     caloric restriction model leads to a reduction in age - associated thymic involution 42 .
obviously , much more work is needed to understand the 
     crosstalk and interregulatory relationships between chemokines and cytokines , mesenchymal cells 
     and adipocytes during the aging process , in particular during the thymic involution process .
adipocytes and preadipocytes secrete a number of inflammatory cytokines including lif , il-1 , 
     il-6 , tnf- among others 43 , 44 . in the thymus , we have observed an increased level of lif , 
     il-6 , tnf- and other pro - inflammatory cytokines in the supernatant of thymus explants 
     of 10 month - old mice as compared to the thymus explants of 2 month - old animals ( our unpublished 
     data ) .
in addition , the expression of lif , oncostatin m and il-6 increases in the thymus with 
     age 8 , 45 .
thus , it 
     is quite reasonable to hypothesize that the production of proinflammatory cytokines by 
     adipocytic cells could actually contribute to oxidative stress in the thymus and , consequently , 
     to the age - associated thymic involution process , killing thymocytes and/or thymic epithelial 
     cells and favoring the lipid accumulation in adipocytic cells .
our observations associating the 
     increased presence of adipocytic / fat - storing mesenchymal cells and loss of thymocytes with 
     advancing age lead to a novel conceptual point of view concerning the understanding of the 
     process of thymic physiology in aging , where the increase in adipocytes an fat - bearing cells may 
     play and active role in thymic tissue loss rather than simply increasing as a consequence of 
     thymic loss by some other as - of - yet undescribed mechanism . based on these findings , studies are 
     underway to investigate the role of adipocytes and adipogenic precursors in the aging thymus 
     physiology as well as their possible contribution to age - associated thymic involution .
these 
     studies may eventually lead to the development of strategic therapies to improve thymic 
     integrity and thymopoiesis during aging .
 age - associated thymic involution is characterized by decreased thymopoiesis , adipocyte 
      deposition and changes in the expression of various thymic microenvironmental factors . in this 
      work 
 , we characterized the distribution of fat - storing cells within the aging thymus . 
 we found 
      an increase of unilocular adipocytes , ertr7 + and ccr5 + fat - storing 
      multilocular cells in the thymic septa and parenchymal regions , thus suggesting that 
      mesenchymal cells could be immigrating and differentiating in the aging thymus . 
 we verified 
      that the expression of ccr5 and its ligands , ccl3 , ccl4 and ccl5 , were increased in the thymus 
      with age . hypothesizing that the increased expression of chemokines and the ccr5 receptor may 
      play a role in adipocyte recruitment and/or differentiation within the aging thymus , we 
      examined the potential role for ccr5 signaling on adipocyte physiology using 3t3-l1 
      pre - adipocyte cell line . 
 increased expression of the adipocyte differentiation markers , 
      ppar2 and ap2 in 3t3-l1 cells was observed under treatment with ccr5 ligands . 
      moreover , 3t3-l1 cells demonstrated an ability to migrate in vitro in response 
      to ccr5 ligands . 
 we believe that the increased presence of fat - storing cells expressing ccr5 
      within the aging thymus strongly suggests that these cells may be an active component of the 
      thymic stromal cell compartment in the physiology of thymic aging . 
 moreover , we found that 
      adipocyte differentiation appear to be influenced by the proinflammatory chemokines , ccl3 , ccl4 
      and ccl5 . 
Introduction
Material and Methods
Results
Discussion