human leukocyte antigen - g ( hla - g ) is a natural molecule involved in the establishment and maintenance of maternal tolerance to semiallogeneic fetal tissues [ 16 ] .
decreased expression of hla - g during pregnancy has been noted as a contributing factor to preeclampsia and multiple miscarriages [ 711 ] . in addition , hla - g expression has been identified in pancreas , adult thymic cells , and stem cells , as well as in pathological conditions including cancer , transplantation , hiv infection , and inflammatory diseases [ 1227 ] .
hla - g binds to several receptors , including ilt2 , ilt4 , and kir2dl4 receptors , to inhibit immune responses of myelomonocytic cells , dendritic cells , t cells , b cells , and nk cells [ 5 , 2835 ] .
in addition to membrane - bound forms ( hla - g1 , -g2 , -g3 , and -g4 ) , hla - g is also presented by several soluble isoforms ( shla - g1 , -g5 , -g6 , and -g7 ) generated through two mechanisms : alternative splicing and proteolytic release , which is known to be mediated by metalloproteases [ 36 , 37 ] .
significantly high levels of shla - g were determined in several physiological and pathological conditions , including an association with higher pregnancy and implantation rates .
it has been determined that a high level of shla - g is correlated with clinical manifestation of several diseases , rheumatoid arthritis , systemic lupus erythematosus , asthma , and hiv infection .
increased levels of shla - g were correlated with disease progression in patients with hematological malignancies and solid tumors , including patients with acute leukemia , lymphoma , chronic lymphatic leukemia , melanoma , breast cancer , glioma , and renal and lung carcinomas [ 25 , 38 ] .
recent studies showed that shla - g molecules are involved in prolongation of allograft survival in patients with organ / tissue transplantation [ 3941 ] . in most studies plasma / serum levels of hla - g was determined by elisa .
however , hla - g - specific elisa has limitations and does not discriminate the presence of monomer or dimer isoforms of hla - g .
new data has revealed that disulfide - linked dimeric complexes of hla - g have high preferential binding to immune inhibitory receptors , induce efficient immune inhibitory receptor signaling , and have strong functional activities [ 32 , 4245 ] .
limited data are available on the role of hla - g dimers in clinical pathological conditions .
the outstanding questions are whether these hla - g dimers are formed in patients with organ transplantation and what their function might be in the prolongation of allograft survival . here
first , we determined that the levels of shla - g dimers were significantly higher in patients with no rejection episode compared with patients that have a chronic rejection of a kidney transplant .
the high levels of shla - g dimers were associated also with increased expression of the membrane - bound form of hla - g on monocytes from patients that have no rejection episode of kidney transplant .
hla - g also has the ability to reduce inflammatory responses through the inhibition of immune cells to produce proinflammatory molecules .
increased expression of mmps was observed in several human diseases , including cancer and autoimmune diseases , suggesting an involvement of these enzymes in immunity , inflammatory responses , and repair mechanisms . mmp-9 and mmp-2 are especially able to modulate inflammatory responses via cytokine / chemokine actions .
here we demonstrated that the high levels of shla - g dimers in kidney transplant patients that have no episodes of rejection were associated with decreased plasma levels of mmp-9 .
we also determined that the increased levels of shla - g dimers linked to the lower levels of proinflammatory cytokines , suggesting the potential role of shla - g dimers in controlling the accompanying inflammatory state . from these findings
, shla - g dimers might be useful as a potential marker to control rejection and the inflammatory status of human kidney allotransplants .
we enrolled kidney transplant recipients in the study , of which 50 had no evidence of rejection ( nr ) and 17 had chronic rejection ( cr ) .
kidney recipients in both categories had similar distribution with respect to gender , age , and race .
the distribution of donor source ( living or deceased ) , cold ischemia time , primary cause of renal failure , and immunosuppressive treatment between each group of recipients was not statistically different .
average creatinine levels of cr patients were significantly higher than nr patients ( p < 0.05 ) .
the protocol was approved by the human assurance committee of georgia regents university , and written informed consent was obtained from all subjects in the study .
pbmcs and red blood cells were isolated from buffy coats using histopaque 1077 ( sigma - aldrich , st .
total protein of plasma samples was measured using the bradford method ( bio - rad , richmond , ca , usa ) . diluted (
1  :  50 ) plasma samples were loaded onto 10% gelatin gels , and electrophoresis was performed at 100 constant voltages .
gels were washed twice with 2.5% triton - x for 20  min and then incubated at 37c overnight in zymography development buffer ( bio - rad ) . the following day , to visualize the bands , gels were stained with coomassie brilliant blue dye ( bio - rad ) for 3  hrs and destained with destaining solution for 45  min .
the gels were rehydrated in water overnight , then scanned and analyzed using imagej program developed at the national institutes of health ( usa ) .
depletion of unwanted proteins that could interfere with the immunoprecipitation of hla - g from plasma was achieved using proteoprep immunoaffinity albumin and igg depletion kit ( sigma - aldrich ) following the manufacturer 's protocol .
100  l of depleted plasma were mixed with 100  l of cold ripa buffer and incubated on ice for 15  min .
20  l of protein g bead slurry was added to the plasma lysate , then incubated at 4c for 60  min and centrifuged at 10,000  g for 10  min . 2  l of mem - g/9 mab ( santa cruz biotechnology , dallas , tx , usa ) was added to the supernatant and the mixture was incubated at 4c overnight . after incubation , 50  l of protein g bead slurry was added to the plasma lysate , incubated at 4c for 1  hr , and centrifuged at 10,000  g for 30  sec .
the mixture was denatured at 95c for 5  min and centrifuged at 10,000  g for 5  min .
30  l of immunoprecipitated plasma was separated on 10% running gel and 5% stacking gel and transferred to pvdf membrane .
the membrane was blocked with 5% bsa and incubated with mem - g/9 primary mab , followed by goat anti - mouse igg - hrp secondary ab ( santa cruz biotechnology ) .
quantification of blotted proteins was determined by densitometry analysis of scanned films using imagej software .
cytokine and chemokine plasma levels were measured using the multi - analyte elisarray kits ( qiagen , valencia , ca , usa ) according to the manufacturer 's recommendations .
the array kits are designed for the simultaneous detection of up to 12 pro- and anti - inflammatory cytokines and chemokines ( il1 , il1 , il2 , il4 , il6 , il8 , il10 , il12 , il17a , ifn , tnf , and gm - csf ) .
pbmcs from both groups of patients ( nr and cr ) were treated with human trustain fcx ( fc receptor blocking solution ; biolegend , san diego , ca , usa ) and stained using fluorochrome - conjugated human - specific mabs against cd3 , cd4 , cd8 , cd14 , cd19 , and hla - g .
all mabs were purchased from bd biosciences ( san jose , ca , usa ) or from biolegend .
cytometry was performed on a cytometer facscanto ( bd , franklin lakes , nj , usa ) and data were analyzed using flowjo software ( tree star inc , ashland , or , usa ) or cell quest software ( bd biosciences ) .
some results are expressed as percentage of positive cells obtained with specific ab compared to irrelevant isotype - matched ab .
statistical analysis was performed using ncss ( ncss llc , kaysville , utah , usa ) and graphpad ( graphpad inc . ,
normality and continuous numeric data was checked using the kolmogorov - smirnoff one - sample test , and comparisons were performed by student 's t test or by mann - whitney u test when appropriate .
in this study we evaluated the levels of monomer and dimer forms of shla - g1 and hla - g5 isoforms in patients with nonrejected ( nr ) and chronic rejected ( cr ) kidney allografts .
immunoprecipitation and western blot analysis of plasma patients resulted in the discovery of two bands ; one corresponding to the expected molecular mass of 39  kda , which represents the shla - g monomer , and one approximately twice that ( shla - g dimer ) ( figure 1(a ) ) .
the supernatant from hla - g5-transfected 721.221 human lymphoblastoid cells was used as a positive control .
both forms of shla - g had been determined in the plasma of nr and cr patients , and there was considerable variation in shla - g levels within each group . however , the level of total shla - g was significantly higher ( p = 0.03 ) in nr compared with cr kidney transplant patients ( figure 1(b ) ) .
in addition , the level of the monomer form of shla - g was slightly higher in the nr group compared with the cr group ( figure 1(c ) ) .
we determined that the dimer form of shla - g was dominant in the plasma of both groups of patients .
moreover , the level of the dimer form of shla - g was significantly elevated ( p = 0.03 ) in the nr group of patients ( figure 1(d ) ) .
these data indicate that the dimer form of shla - g is present and dominates in the plasma of kidney transplant patients .
it is known that shla - g proteins can be generated by two mechanisms : alternative splicing and proteolytic release , which is mediated by metalloproteases . to determine the potential contribution of the membrane - bound form of hla - g1 shedding into the pool of shla - g molecules in plasma patients ,
the expression of hla - g1 on the cell surface of peripheral monocytes and t and b cells has been analyzed in both groups of patients .
there was no significant difference in the number of hla - g1-positive t cells and b cells between nr and cr patients ( figures 2(a ) and 2(d ) and data not shown ) .
overall , in both groups of patients , hla - g1-positive cells represent a small fraction ( 26% of the total ) of t and b cells . as expected , the majority of hla - g1-positive cells in the peripheral blood of both nr and cr patients were determined in the population of monocytes ( figures 2(a ) , 2(b ) , and 2(c ) ) . however , the number of hla - g1-positive monocytes was significantly elevated ( p = 0.002 ) in the nr patients , but not in the cr patients ( figure 2(e ) ) .
these data revealed that the increased expression of hla - g1 on monocytes from nr patients might have a substantial contribution to the elevated plasma levels of shla - g monomer and dimer forms in those patients .
since one of the mechanisms of the production of shla - g involves shedding of the membrane - bound form of hla - g1 by metalloproteases and since mmps play a crucial role this class of enzymes , we investigated the levels of mmp-2 and mmp-9 in the plasma of kidney transplant patients .
zymography analysis of transplant patients showed the presence of two bands at 68  kda and 90  kda , which correspond to mmp-2 and mmp-9 , respectively ( figure 3(a ) ) .
as shown in figure 3(a ) , plasma from both groups of patients contains substantial amounts of mmp-2 and mmp-9 , with no significant difference in the plasma levels of mmp-2 and mmp-9 between nr and cr patients ( figures 3(c ) and 3(d ) ) .
however , a tendency toward elevation of mmp-9 levels was observed in the cr kidney transplant patients ( figure 3(d ) ) .
since plasma from both the nr and cr groups of patients contains shla - g , this data additionally support the possibility that mmps might play a role in contributing to the pool of total shla - g by shedding the membrane - bound hla - g1 molecules .
since hla - g , mmp-2 , and mmp-9 are all involved in regulation of the inflammatory response by modulation of cytokines and chemokines and the inflammatory response represents a critical stage in rejection or survival of allogeneic transplants , we next determined the levels of proinflammatory cytokines in kidney transplant patients .
we thus investigated whether the plasma level of proinflammatory cytokines differs between nr and cr kidney transplant patients .
for this purpose , we have used a multi - analyte elisarray kits designed to simultaneously assess the levels of 12 pro- and anti - inflammatory cytokines and chemokines ( il1 , il1 , il2 , il4 , il6 , il8 , il10 , il12 , il17a , ifn , tnf , and gm - csf ) .
there was considerable variation in cytokines levels within both the nr and cr patients , especially for il-1 , il-4 , il-12 , and ifn- ( figure 4 ) .
however , the cr kidney transplant patients had significantly elevated levels of proinflammatory cytokines il-2 ( p = 0.005 ) , il-1  ( p = 0.05 ) , and il-6  ( p = 0.05 ) ( figure 4 ) .
these data support our observation that the dimer form of shla - g associates with control of inflammatory responses in kidney transplants patients .
hla - g is natural molecule involved in the establishment and maintenance of maternal tolerance to fetal semiallogeneic tissues .
hla - g binds to several immune cell inhibitory receptors , for example , ilt2 , ilt3 , and ilt4 , to downmodulate immune responses of myelomonocytic cells , t cells , b cells , and nk cells .
limited polymorphisms , restricted tissue expression , and a relatively restricted peptide presentation make hla - g a unique molecule , unlike the classical hla class i molecules .
recently , another unusual characteristic of hla - g has been discovered : its ability to form a disulfide - linked dimer form both in solution and at the cell surface .
hla - g , unlike most other mhc class i molecules , has two free cysteine residues located in positions 42 and 147 in extracellular domains 1 and 2 , respectively .
hla - g molecules refolded in vitro form a disulfide - linked dimer with an intermolecular cys42-cys42 disulfide bond .
soluble hla - g5 molecules expressed by human 293 t cells also form disulfide - linked dimeric and additional oligomeric forms , which can reduce the level of cd8 expression on cytotoxic t lymphocytes ( ctls ) . the efficiency of inhibitory signaling is dependent upon several factors , including the stability and avidity of the ligand and its proper structural orientation , which significantly affects the affinity and signaling to targeted inhibitory receptors .
mutagenesis studies of the free cysteines suggested that the hla - g dimer more efficiently inhibits nk killing than the monomer and increases the efficiency of ilt2 signaling [ 32 , 42 , 43 , 48 ] .
experimental data from several groups , including our laboratory , suggest that hla - g dimer has increased avidity and proper structural orientation to induce efficient inhibitory signaling during ligation with human ilt2 and ilt4 , and murine pir - b - inhibitory receptors [ 32 , 44 , 45 ] .
this makes hla - g dimer as the most powerful ligand form for modulation of inflammatory and alloimmune responses in several pathological conditions , including the prolongation of kidney allograft survival or graft acceptance .
many studies have been designed to determine shla - g in the plasma or serum of patients suffering from various diseases .
shla - g levels were determined in spontaneous miscarriages , in autoimmune diseases , in solid organ transplantation , and in various malignancies . in almost all the studies ,
unfortunately , the available shla - g elisa determines the total amount of shla - g protein only , which includes both monomer and dimer forms .
since the dimer represents the most powerful form of shla - g , it is very critical to analyze the levels of shla - g dimer in healthy and disease conditions . to date
, no study has investigated whether hla - g disulfide - linked homodimers are present in plasma from kidney transplant patients . here we show that shla - g dimers are present in plasma from kidney transplant patients . the levels of shla - g dimer were significantly elevated in patients with no rejection episodes compared with patients with chronic rejection , indicating the association of shla - g dimers with the prolongation of kidney allograft survival . in support of that , similar levels of expression and percent of immune inhibitory receptor ilt2- and ilt4-positive cells has been determined on monocytes and t and b cells in both groups of patients ( data not shown ) .
this clinical finding is in agreement with our previous study using animal models demonstrating that hla - g dimers prolong the survival of allogeneic skin transplants in ilt transgenic mice ( [ 32 , 49 , 50 ] and unpublished data ) . in the future
, it will be important to determine the percentages of shla - g1 and shla - g5 dimers in total shla - g .
however , to date , there is no data available demonstrating that shla - g1 and shla - g5 dimers have different binding and/or different efficiency to induce inhibitory receptor signaling .
our results show that the number of hla - g1-positive monocytes is significantly increased in nr patients , indicating that the percent of shla - g1 dimer might have been elevated within the total level of shla - g dimers in plasma of the patients with no episodes of rejection .
recently , rizzo et al .   demonstrated an effective link between mmp-2 and hla - g1 shedding in 721.221-g transfected cell line , jeg3 cell line , and il-10-treated pbmcs from five healthy donors using an in vitro experiments .
however , the process of shedding and especially dimerization of shla - g1 in vivo and in pathological conditions , including kidney transplant patients , could be affected by several factors and requires additional investigation .
the anti - inflammatory effects of shla - g dimers represent a new finding for this form of hla - g .
recently published data by kuroki et al .   demonstrating that in an animal model of collagen - induced arthritis , hla - g dimer interacting through a murine ilt homologue , the pir - b receptor exhibited significantly more anti - inflammatory effects compared to monomer . to date , no data is available on studies of the anti - inflammatory effect of shla - g dimers in clinical applications .
we demonstrate here that an increased level of shla - g dimers in kidney transplant patients with no rejection episodes is linked to significantly lower levels of proinflammatory cytokines il-2 , il-1 , and il-6
. it will be important to dissect the mechanisms of shla - g dimers controlling inflammatory responses .
the anti - inflammatory effect of shla - g dimers opens a new strategy to generate useful agents to control inflammatory responses with minimal side effects . in conclusion
, our study shows that shla - g dimers are associated with better survival of kidney allografts and control of the accompanying inflammatory response in kidney transplant patients .
thus shla - g dimers can be a potential biomarker to control human alloimmune and inflammatory responses .
 human leukocyte antigen - g ( hla - g ) contributes to acceptance of allografts in solid organ / tissue transplantation . 
 most studies have determined that soluble hla - g isoforms are systematically detected in serum / plasma of transplanted patients with significantly fewer episodes of acute and/or chronic rejection of allogeneic tissue / organ . 
 current models of the interactions of hla - g and its specific receptors explain it as functioning in a monomeric form . 
 however , in recent years , new data has revealed the ability of hla - g to form disulfide - linked dimeric complexes with high preferential binding and functional activities . 
 limited data are available on the role of soluble hla - g dimers in clinical pathological conditions . 
 we describe here the presence of soluble hla - g dimers in kidney transplant patients . 
 our study showed that a high level of hla - g dimers in plasma and increased expression of the membrane - bound form of hla - g on monocytes are associated with prolongation of kidney allograft survival . 
 we also determined that the presence of soluble hla - g dimers links to the lower levels of proinflammatory cytokines , suggesting a potential role of hla - g dimers in controlling the accompanying inflammatory state . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion