systemic lupus erythematosus ( sle ) is a systemic autoimmune disease with manifestations in multiorgans that are induced by the deposition of circulating autoantibody - autoantigen complexes ( immune complexes , ic ) and amplified by subsequent infiltration of different types of leukocytes promoting the inflammation .
lupus nephritis ( ln ) , a major cause of morbidity and mortality in up to 60% sle patients , is characterized by inflammation of the kidney .
ic and subsequent complement activation both induce the activation and damage of renal cells that further release inflammatory factors leading to the infiltration of leukocytes into glomerular , tubulointerstitial , and perivascular regions of the inflamed kidney to amplify the renal inflammation and damage .
therefore , leukocyte recruitment to the inflamed kidney is a critical step in the development of ln .
chemokines are a group of cytokines with small molecular weight whose main action is the recruitment of leukocyte subsets under homeostatic and pathological conditions . through interacting with chemokine receptors that are expressed on the cell surface as 7-transmembrane proteins coupled with g - protein for signaling transduction , chemokines can induce firm adhesion of targeted cells to the endothelium and direct the movement of targeted cells to their destination according to the concentration gradient of a given chemokine . through this mechanism chemokines
chemokines are classified into four subfamilies according to the first two cysteines and the amino acid residues in between at n - terminal end of the polypeptide . based on whether the first two cysteines are adjacent , separated by one residue , or separated by three residues , a chemokine is classified into ccl , cxcl , or cx3cl family , respectively .
chemokine receptors are named corresponding to the subfamilies of chemokines as ccr , cxcr , cx3cr , or xcr , respectively .
individual chemokines and chemokine receptors discovered to date have been reviewed elsewhere with summarized tables [ 58 ] .
homeostatic chemokines and chemokine receptors are those important for the homing of progenitor cells and mature immune cells into the primary / secondary immune tissues for the development of the immune system and into peripheral nonimmune tissues for the tissue - specific functions and immune surveillance [ 5 , 9 ] .
examples are ccr7 that is expressed on nave lymphocytes and dendritic cells for recruitment into lymph nodes by ccl19 and ccl21 as part of the normal immune system development ; cxcl12 that is important for the retention of cxcr4 hematopoietic stem cells ( hscs ) in hsc niches in the bone marrow ; ccl2 that is critical for ccr2 monocytes emigration from the bone marrow ; and cx3cl1 that is essential for cx3cr1 monocytes patrolling along the blood vessels . on the other hand ,
when there is an infection or injury - induced inflammation , activated immune cells will upregulate some chemokine receptors and migrate into inflamed immune and nonimmune tissues by recognizing correspondingly increased inflammatory chemokines .
for example , upon the stimulation of pathogen - associated molecular pattern molecules ( pamp ) or danger - associated molecular pattern molecules ( damp ) , resident mast cells , macrophages , and dendritic cells will release cytokine signals to induce the upregulation of several inflammatory chemokines expressed by activated endothelial and epithelial cells , such as ccl2 , ccl3 , ccl4 , ccl5 , cxcl1 , cxcl2 , cxcl3 , cxcl5 , and cxcl8 .
consequently , circulating immune cells such as neutrophils , monocytes , and effector t cells will migrate into inflamed tissues using related chemokine receptors such as ccr2 , ccr1 , and cxcr2 .
chemokines , unlike adhesion molecules broadly shared by different types of immune cells , are selectively used by specific cell populations and have been found to be involved in the migration of leukocytes to nephritic kidney of both sle patients and lupus - prone mice .
studies have shown that several immune cell populations are accumulated in the kidney in ln , including various t cell subsets , b cells , plasma cells , nk cells , monocytes / macrophages , dendritic cells ( dc ) , and neutrophils [ 10 , 11 ] . in this review ,
we summarize recent studies on the chemokines that are increased in the kidney as ln progresses and the corresponding chemokine receptors used by renal - infiltrating leukocytes in response to the chemokines .
we focus on those highlighted by multiple studies , including the chemokines cxcl13 , cxcl12 , cxcl9 , cxcl10 , cxcl11 , ccl2 , ccl3 , ccl4 , ccl5 , and cx3cl1 and chemokine receptors cxcr5 , cxcr4 , cxcr3 , ccr1 , ccr2 , ccr5 , and cx3cr1 .
inflammatory factors inducing the expression of chemokines , as well as chemokines that may be used as biomarkers for the diagnosis of ln , are also discussed .
some chemokines are more commonly related to ln than others based on both studies of lupus - prone mouse models and sle patients , which have been summarized in tables 1 and 2 , respectively . to interact with specific chemokine receptors expressed on particular cell populations , these chemokines have diverse biological effects by influencing the migration of different cell populations in both healthy and disease situations . in ln disease , animal model studies suggest that these chemokines contribute to systemic autoimmune responses in immune tissues  thus indirectly promoting ln  and are involved in local renal inflammation with direct effects .
evidence from human studies , on the other hand , suggests the clinical involvement of these chemokines in the development of ln . in the following sections we will discuss each of these ln - related chemokines regarding their biological effects , roles in mouse models of ln , and clinical evidence from studies of lupus nephritic patients .
cxcl13 , also known as b cell - attracting chemokine 1 ( bca1 ) or b lymphocyte chemoattractant ( blc ) , is the chemokine recognized by cxcr5 .
cxcl13 is important in directing the trafficking of cxcr5 cells , including b cells , follicular helper cd4 t cells ( tfh ) , cxcr5cd8 t cells , and cxcr5 dc , all involved in humoral immune responses [ 1214 ] . both b cells and tfh critical for the formation of germinal centers ( gc ) depend on cxcr5 to migrate into the b cell follicles in secondary immune tissues [ 12 , 1517 ] . circulating cxcr5 central or effector memory - like tfh
have also been discovered that could migrate to and function in immune and nonimmune tissues [ 1821 ] . besides tfh , cxcr5cd8 t cells and cxcr5 dc are also found to facilitate b cell responses [ 13 , 14 ] .
therefore , by attracting different types of cxcr5 immune cells , cxcl13 contributes to b cell responses , especially the generation of high affinity antibody - producing cells in gc . in two
commonly used lupus - prone mouse models , nzb / w f1 and mrl / lpr , transcript levels of renal cxcl13 and cxcr5 are consistently increased in aged lupus nephritic mice compared to nonlupus control mice or young mice prior to disease onset [ 2225 ] , suggesting their involvement in the development of ln .
renal macrophages and dc in lupus - prone mice may be the source of cxcl13 in the nephritic kidney [ 24 , 2629 ] , leading to increased migration of cxcr5 b cells and tfh - like cells into the inflamed kidney towards cxcl13 [ 24 , 26 , 30 ] .
further studies with nzb / w f1 mice have shown that , among b cell populations , b1 cells compared to b2 cells express much higher cxcr5 and migrate towards cxcl13 more efficiently in vitro [ 22 , 30 ] , and preferentially migrate into the kidney and lung of diseased mice instead of lymphoid tissues . functionally , b1 cells isolated from nzb / w f1 mice , but not b2 cells , can activate t cells in allogeneic mixed lymphocyte reaction .
cxcr5 cd4 t cells , on the other hand , have been shown to promote igg production from b1 cells in vitro , suggesting potential interaction of b1 and t cells in situ in the nephritic kidney .
however , another study found most renal - infiltrating b cells to be non - class - switched b2 cells in nzb / w f1 mice , leaving the role of renal cxcr5 b in ln an open question . while further studies are required
, these results suggest important functions of cxcr5 and its ligand in the development of ln . the critical roles of cxcl13 and cxcr5 cells in the pathogenesis of ln are also evidenced by studies of anti - cxcl13 neutralizing antibodies in mrl / lpr lupus - prone mice and cxcr5-deficiency in b6/lpr lupus - prone mice [ 31 , 32 ] .
renal pathology , including proteinuria and serum creatinine levels , glomerular and perivascular scores , deposition of ic and complement c3 , and renal il-1 , il-6 , il-33 , and il-17 protein levels , was significantly lower in the neutralizing antibody - treated mice than controls .
systemic autoimmune responses such as the level of circulating anti - double stranded dna ( anti - dsdna ) antibodies and the ratio of splenic th17/treg were reduced as well , suggesting that the pathogenic role of cxcl13/cxcr5 may not be kidney - specific . as the administration of anti - cxcl13 neutralizing antibodies is not tissue - specific , it is difficult to say if reduced renal pathology is due to the secondary effect of decreased systemic autoimmune responses or the direct effect of blocking cxcl13/cxcr5 signal in the kidney .
similar to the cxcl13 blockade study , cxcr5-knockout in b6/lpr mice also downregulated systemic autoimmune reactions , including reduced lymphadenopathy and splenomegaly with reduced gc , b cells , plasma cells , and double negative ( dn ) t cells in secondary lymphoid organs , as well as reduced circulating igg .
importantly , this study also showed reduced infiltration of adoptively transferred dn t cells from cxcr5-deficient b6/lpr mice compared to wild type b6/lpr mice into the kidney of rag1 recipient mice , indicating direct contribution of cxcl13/cxcr5 signal to local renal inflammation in ln .
therefore , cxcl13/cxcr5 contributes to the development of ln both systemically in immune tissues and locally in the kidney of lupus - prone mice .
evidence from the studies of sle patients with ln further suggests the clinical involvement of cxcl13/cxcr5 in the development of ln . in sle patients with ln , but not in healthy controls ( hc ) , cxcl13 and cxcr5 are highly expressed in the cortex of the kidney .
in addition , b cells and tfh - like cells that express cxcr5 have been indicated to infiltrate the nephritic kidney of sle patients and are colocalized with cxcl13-expressing regions .
besides chemoattractant functions , cxcl13 may also contribute to ln by activating cxcr5 renal nonimmune cells such as human podocytes to produce proinflammatory molecules .
cxcl12 , also known as stromal cell - derived factor 1 ( sdf-1 ) , is the ligand of chemokine receptor cxcr4 .
it is involved in the homing , retaining , and survival of cxcr4 hematopoietic stem cells , b cell precursors , and plasma cells in the bone marrow .
in addition , cxcl12 maintains the homeostasis of neutrophils , t cells , and b cells in immune and nonimmune tissues [ 3638 ] .
studies have shown possible involvement of cxcl12 and cxcr4 in ln development . in lupus - prone mouse models ,
cxcr4 is consistently increased in various immune cell populations including b cells , plasma cells , t cells , neutrophils , and monocytes in the circulation and spleen of diseased mice , suggesting enhanced chemotaxis of these cells towards cxcl12 [ 3941 ] .
importantly , both cxcl12 and cxcr4 expressions are increased in the kidney of diseased lupus - prone mice , indicating migration of cxcr4 cells into the kidney as ln progresses [ 23 , 24 , 40 , 42 ] . in particular , studies have shown the increased accumulation of cxcr4 cells in the kidney of diseased mice , including plasma cells , foxp3 cd4 regulatory t ( treg ) cells , foxp3 cd4 conventional t cells , and inflammatory monocytes and neutrophils [ 40 , 41 , 43 ] .
besides promoting leukocyte infiltration , cxcl12/cxcr4 may also deteriorate ln by directly affecting renal tissue cells .
studies have shown that activated parietal epithelial cells ( pecs ) as glomerular progenitor cells are involved in proliferative glomerulonephritis .
normally , pecs possess regenerative potential for the repair of injured kidney [ 45 , 46 ] .
however , in glomerulonephritis , cxcr4 is overexpressed on pecs upon inflammatory stimulation , whereas autoantibodies and inflammatory mediators stimulate cxcl12 production on injured podocytes [ 42 , 44 , 47 ] . consequently , through the interaction between cxcl12 and cxcr4 , pecs migrate into the glomerular tuft during the development of ln , where they predominately form hyperplastic lesions in proliferative glomerulonephritis and lead to glomerulosclerosis by secreting extracellular matrix [ 3 , 44 ] . blocking the interaction of cxcl12/cxcr4 in lupus - prone mice reveals their contributions to both systemic autoimmune responses in secondary lymphoid organs and local renal inflammation .
administration of anti - cxcl12 neutralizing antibodies in nzb / w f1 mice led to increased survival rate and reduced renal inflammation including decreased proteinuria and igg deposition . this may be at least partially due to decreased systemic autoimmune reactions , since circulating anti - dsdna igg and b1a subset in the peritoneal cavity and the spleen were reduced , as well as reduced activated cd4 t cells in the spleen and lymph nodes .
the direct role of cxcl12/cxcr4 network to recruit immune cells in the lupus nephritic kidney is demonstrated in another study with administration of a cxcr4 antagonist in b6.sleyaa lupus - prone mice .
similar to anti - cxcl12 neutralizing antibodies , cxcr4 antagonist ameliorated ln with decreased renal pathological scores and proteinuria and prolonged lifespan .
early administration before severe proteinuria also led to reduced splenomegaly and circulating ana igg , suggesting a systemic effect .
splenic monocytes , activated t cells , and b cells in marginal zone and follicular and germinal center were similarly reduced .
however , late administration after the onset of severe proteinuria did not influence systemic autoimmune responses but led to reduced infiltration of monocytes , neutrophils , and cd4 t cells into the kidney , suggesting a direct effect of cxcl12/cxcr4 in the kidney . in patients with ln , it has been consistently demonstrated that cxcl12 expression is significantly increased in tubules and glomeruli of the kidney , while most circulating cd4 t cells and b cells express cxcr4 in sle patients [ 6265 ] .
although it is debatable whether its level on b cells is reduced   or increased [ 62 , 65 ] , cxcr4-expressing b cells are found to be accumulated in the renal biopsy samples of patients with ln , suggesting involvement of cxcl12/cxcr4 in the kidney of patients with ln .
cxcr3 is a chemokine receptor interacting with three interferon - inducible chemokines , cxcl9 ( monokine induced by gamma - interferon , mig ) , cxcl10 ( interferon - induced protein of 10  kda , ip-10 ) , and cxcl11 ( interferon - inducible t cell alpha chemoattractant , i - tac ) .
several immune cell populations have been reported to express cxcr3 , including nk cells , plasmacytoid dc ( pdc ) , conventional dc ( cdc ) , b cells , and activated t cells . among activated t cells , t helper 1 ( th1 ) cells and effector cd8 t cells
t helper 17 ( th17 ) cells have also been reported to express cxcr3 , although ccr6 is the predominant chemokine receptor on their surface . since the expression of cxcr3
is induced upon activation of immune cells , especially in effector t cell populations , activated immune cells can migrate into inflamed peripheral tissues where cxcr3 ligands are induced .
the three cxcr3 ligands under different circumstances have shown redundancy , dominance , collaboration , or antagonism to one another .
therefore , cxcr3 and its ligands are mainly associated with the effector stage of immune response and are regulated in a more complex fashion than single paired chemokines / chemokine receptors .
cxcr3 and its ligands are involved in the pathogenesis of sle . in lupus - prone mice ,
most commonly nzb / w f1 mice and mrl / lpr mice , cxcr3-expressing t cells and plasma cells as activated effector populations in the secondary lymphoid organs are increased during the development of ln [ 41 , 48 , 66 , 74 ] .
importantly , studies have shown that cxcr3 and its ligands are increased in the nephritic kidney of lupus - prone mice , suggesting migration of cxcr3-expressing effector cells from the secondary lymphoid organs into the inflamed kidney [ 23 , 24 , 49 , 66 , 7476 ] .
detailed studies with mrl / lpr and nzb / w f1 mice reveal that cxcr3 is expressed on different renal - infiltrating cells with varied proportions , including cd4 t cells ( 1533% ) , cd8 t cells ( 1033% ) , b220 cells ( including both b cells and pdc , 25% ) , plasma cells ( 40% ) , and macrophages ( 5% ) [ 23 , 48 , 49 ] .
all renal cxcr3 t cells are confirmed as cd44 activated t cells , while cd44 nave t cells are cxcr3-negative .
in addition , renal - infiltrating cxcr3 plasma cells can secrete igg instead of igm , indicating their pathogenic role in promoting ln .
while both cxcr3 and its ligands are increased in the kidney of lupus - prone mice with ln , their deficiencies in lupus - prone mice have shown inconsistent or even contradictory results .
however , cxcr3- or cxcl9-deficiency in the nephrotoxic serum nephritis ( nsn ) model showed reduced nephritic disease with decreased igg deposits and activated t cells and macrophages in the kidney .
this suggests that cxcl9 rather than cxcl10 may be critical for cxcr3-dependent cellular infiltration of the kidney in ln .
consistent with this , another study showed that cxcl9 in the kidney of diseased mrl / lpr mice was the most abundant chemokine for t cell trafficking .
however , circulating antigen - specific igg was also reduced in cxcr3- or cxcl9-deficient nsn model , suggesting that cxcr3/cxcl9 interaction may influence systemic immune responses and indirectly affect kidney pathology .
further studies with cxcr3-knockout mrl / lpr and nzb / w f1 mice have shown different effects on the development of ln .
with cxcr3-deficiency in mrl / lpr mice , glomerular pathology score was reduced with decreased t cells and macrophages infiltration around glomeruli , ameliorated renal lesion , and decreased proteinuria .
ifn-producing t cells and il-17-producing t cells were also reduced in the kidney but not in the spleen or lymph nodes of cxcr3-knockout mrl / lpr mice .
importantly , renal igg and c3 deposits and circulating total igg and anti - dsdna igg were not different between cxcr3-knockout and wild type mrl / lpr mice , suggesting a direct effect of cxcr3 and its ligands on the kidney by recruiting activated effector t cells and macrophages .
however , in nzb / w f1 mice , cxcr3 deficiency did not change either the infiltration of plasma cells and t cells to the kidney or the course of ln .
therefore , further studies are required to determine whether cxcr3 is important for ln development and which ligand(s ) are critical for the infiltration of cxcr3 cells to the kidney of lupus - prone mice . despite controversial results from studies of lupus - prone mice ,
evidence from sle patients still suggests the possible involvement of cxcr3 and its chemokine ligands in the development of ln .
patients with active sle compared to hc or patients with inactive disease have reduced cxcr3 cd4 t cells in the circulation , suggesting infiltration of the cells into peripheral tissues .
in addition , several studies have shown that , in sle patients with ln , compared to hc or patients without nephritic involvement , cxcr3 cells ( mostly t cells ) are increased in the kidney and urine , which is correlated with increased expression of renal cxcr3 ligands [ 26 , 66 , 67 ] . moreover
, it has been found that cxcr3 cells are accumulated in tubulointerstitial regions and around glomeruli in the kidney of lupus nephritic patients , account for 60% of total infiltrating cells , and are positively correlated with proteinuria . among the three cxcr3 ligands ,
cxcl10 is most increased in sle patients and localized in the same region as cxcr3 cells in the nephritic kidney .
besides cxcr3-expressing t cells , a group of pathogenic cd19 b cells also express cxcr3 at a high level in sle patients and migrate towards cxcl9 in vitro , suggesting their potential to migrate into inflamed peripheral tissues such as the kidney .
ccr1 and ccr5 share the same ligands , ccl3 ( macrophage inflammatory protein 1-alpha , mip-1 ) and ccl5 ( regulated upon activation , normally t - expressed , and presumably secreted , rantes ) .
ccr1 is expressed on cd34 bone marrow progenitor cells , monocytes , nk cells , t cells , and preferentially on cd45ro activated / memory t cells [ 50 , 77 ] .
ccr5 is expressed on monocytes / macrophages and t cells ( both cd4 and cd8 subsets ) , especially on th1 cells [ 50 , 79 ] .
interestingly , monocytes express a high level of ccr1 but low ccr5 , while the expression pattern is the opposite in activated / memory t cells , suggesting selective expression of ccr1 and ccr5 in monocytes and activated t cells , respectively .
ccr1 or ccr5 knockout mice have been developed to study their functions in different disease models .
even though ccr1 and ccr5 share the same chemokine ligands , studies with unilateral ureteral obstruction and renal ischemia - reperfusion injury models have shown that ccr1 but not ccr5 is essential for t cells , macrophages , and neutrophils infiltration in the tubulointerstitial region of the kidney [ 8183 ] .
moreover , ccr1-deficient mice have enhanced macrophage and t cell infiltration to the glomerular region of the kidney in a nephrotoxic nephritis model , suggesting that such infiltration is ccr1-independent .
ccr1-deficient mice also exhibit increased circulating antigen - specific , th1-biased , pathogenic igg2a response , indicating that ccr1 is also involved in th1-dependent systemic humoral immune response .
however , in a host versus graft disease ( hvgd ) model , ccr1-deficiency shows a protective effect by inhibiting chronic cardiac allograft rejection , which makes the role of ccr1 complicated in different diseases possibly depending on whether humoral immune responses are involved and/or which tissues and immune cell populations are involved . regarding ccr5 deficiency ,
macrophages from ccr5-knockout mice have reduced ability to produce inflammatory cytokines including il-6 , il-1 , and tnf , rendering defective bacteria clearance in a listeria monocytogenes infection model .
ccr5-deficient t cells , on the other hand , have elevated production of ifn , granulocyte macrophage colony - stimulating factor ( gm - csf ) , and il-4 with enhanced delayed - type hypersensitivity reaction and humoral immune responses following antigen challenge in ccr5-deficient mice .
ccr5 also contributes to the recruitment of treg in lymphoid and nonlymphoid tissues , which is important in suppressing effector responses in graft versus host disease- ( gvhd- ) targeted organs .
therefore , ccr5 deficiency in different diseases leads to different outcomes depending on which cell types are critical and whether the initial immune response ( in lymphoid organs ) or the effector phase ( in nonimmune tissues ) is involved . in lupus - prone mice ,
ccr1 , ccr5 , and their ligands are increased in the kidney during ln development [ 24 , 25 , 28 , 4952 , 54 , 75 , 76 ] .
studies have shown that , in nephritic nzb / w f1 mice , both renal t cells and monocytes / macrophages have elevated ccr1 expression on the surface . in mrl / lpr mice ,
the administration of a ccr1 antagonist at late stage improved ln with reduced interstitial lesions including decreased infiltration of t cells and monocytes / macrophages , reduced inflammation - induced proliferating and apoptotic cells , and reduction of tubular atrophy and interstitial fibrosis .
however , glomerular igg deposits and different isotypes of circulating anti - dsdna igg reflecting systemic humoral autoimmune response did not change , suggesting a direct effect of ccr1 antagonism on preventing renal infiltration of t cells and macrophages .
this was confirmed by reduced renal infiltration of adoptively transferred t cells and macrophages pretreated with the ccr1 antagonist .
the role of ccr1 in ln was limited in interstitial region , as glomerular injury and proteinuria were not improved by ccr1-antagonist administration in mrl / lpr mice . in nzb / w f1 mice , the effect of ccr1 antagonist administration at late stage has also been studied . besides the similar effects of ccr1 blocking t cells and macrophage infiltration , the study with nzb / w f1 mice also showed prolonged lifespan and improved glomerular injury including reduced proteinuria . in mrl / lpr mice ,
the extent of ccr5 expression is debated , as over 50% of renal t cells express ccr5 in one study , whereas only 1% of t cells are shown to express ccr5 in another study [ 49 , 52 ] .
renal - infiltrating macrophages , on the other hand , are ccr5-positive in mrl / lpr mice .
contrary to the effects of ccr1 blocking , ccr5 knockout in mrl / lpr mice deteriorated ln with increased proteinuria and tubulointerstitial infiltration of total cd3 t cells and f4/80 macrophages in the kidney .
foxp3 tregs were also increased in the kidney of ccr5-knockout mrl / lpr mice , but ln progression was not reversed by the increase of treg cells .
systemic humoral immune responses were not affected by ccr5 deficiency , as the circulating anti - dsdna igg and renal igg / c3 deposits were not different between ccr5-knockout and wild type mrl / lpr mice . however , ccr5-knockout mrl / lpr mice exhibited increased splenomegaly and elevated circulating / renal ccl3 , suggesting that renal - infiltrating immune cells may use alternative chemokine receptors responding to ccl3 such as ccr1 to migrate into the kidney and promote ln .
this study reveals possible roles of ccr5 in negatively regulating ln progression by modulating ccl3 production and controlling lymphoproliferation in the spleen .
therefore , it appears that ccr1 and ccr5 may , respectively , promote or attenuate the development of ln in lupus - prone mice . in sle patients ,
ccr1 , ccr5 , and their ligands are also increased in the kidney during the development of ln [ 69 , 70 , 86 , 87 ] .
evidence from sle patients further shows that most ccr1 cells infiltrating in the kidney are cd68 macrophages [ 63 , 69 ] , while ccr5 , on the other hand , is expressed on both circulating and renal - infiltrating t cells in sle patients , particularly interstitial infiltrating t cells .
ccr2 is expressed on a fraction of monocytes , dendritic cells , nk cells , and t cells , and one of its ligand is ccl2 ( monocyte chemoattractant protein-1 , mcp-1 ) [ 50 , 88 ] .
ccr2 expression on monocytes is important for both their egression from bone marrow and extravasation into inflamed tissues . besides chemoattractant function for monocytes , ccr2 and ccl2
t cells from ccr2-deficient mice produce less ifn upon stimulation , while ccl2 is associated with th2 cell polarization and enhances il-4 production by t cells . in lupus - prone mice , ccr2 and ccl2 are increased in the kidney during the development of ln , suggesting the recruitment of ccr2 leukocytes into the inflamed kidney by ccl2 [ 24 , 25 , 5456 , 89 ] . using mrl / lpr mice
, studies have shown that most renal - infiltrating ccr2 cells are macrophages and not t cells [ 52 , 54 ] .
in addition , ccl2 is mainly expressed in the tubulointerstitial regions of the kidney in lupus - prone mice [ 55 , 89 ] . by blocking the interaction between ccr2 and ccl2 in mrl / lpr lupus - prone mice
, studies have shown that ccl2/ccr2 network contributes to ln development through both systemic and local mechanisms . in both ccl2/ccr2 antagonist experiments and ccl2/ccr2-knockout models ,
animal lifespan was consistently prolonged with reduced ln including less glomerular and tubulointerstitial infiltration of t cells and macrophages , although severe proteinuria in old mice was not improved [ 5559 ] .
in addition , the pathology and inflammation in the lung and skin of ccl2/ccr2-deficient mrl / lpr mice were reduced , suggesting the systemic involvement of ccl2/ccr2 in multiperipheral tissues . by further comparing the differences between antagonist and knockout models
, it was evident that ccl2/ccr2 antagonists did not improve splenomegaly , lymphadenopathy , and circulating total / autoantibodies , suggesting the local involvement of ccl2/ccr2 in autoimmune target tissues , such as the kidney [ 56 , 58 , 59 ] . in contrast , ccl2/ccr2-knockout mrl / lpr mice exhibited reduced circulating anti - dsdna igg , diminished lymphadenopathy , and decreased percentage of circulating cd8 t cells , suggesting ccl2/ccr2 network also contributes to systemic autoimmune reactions in the immune tissues , through which ln progression was indirectly promoted [ 55 , 57 ] .
interestingly , anti - ccl2 spiegelmer , a ccl2 antagonist , blocked the emigration of monocytes from the bone marrow of mrl / lpr mice , which suggested an additional mechanism of how ccl2/ccr2 may promote ln by facilitating monocytes migration from the bone marrow into the kidney .
together , these results suggest the importance of ccr2 and ccl2 in promoting ln . in sle patients , ccr2 and ccl2 expression
same as shown in lupus - prone mice , ccl2 is mainly expressed in the tubulointerstitial region of the kidney in sle patients .
renal endothelial cells , epithelial cells , and infiltrating leukocytes could be the source of ccl2 . in patients with active sle , a small proportion of t cells ( both cd4 and cd8 ) express ccr2 and are reduced in the blood circulation , suggesting their migration from the blood to inflamed peripheral tissues such as the kidney . cx3cl1 , also known as fractalkine , is the only chemokine with cx3c - motif discovered to date that interacts with its unique chemokine receptor , cx3cr1 .
cx3cr1 is expressed on a fraction of monocytes / macrophages , dendritic cells , nk cells , t cells , and particularly cd8 cytotoxic t cells [ 9093 ] .
different from other chemokines , cx3cl1 possesses a soluble form and a transmembrane form , which function to induce chemotaxis and adhesion of cx3cr1 leukocytes , respectively .
cx3cr1/cx3cl1 interaction also provides antiapoptotic signals to sustain the survival of cx3cr1 leukocytes [ 50 , 94 , 95 ] .
studies have shown possible involvement of cx3cl1 and cx3cr1 leukocytes in the development of ln . in mrl / lpr mice ,
cd16 cells in glomeruli are increased with lupus development , with increased protein level of cx3cl1 detectable in glomeruli , interstitial microvasculature , and arterial regions . unlike mrl / lpr mice , cx3cr1 and cx3cl1 expression in the kidney of nzb / w f1 mice
do not change with lupus progression , suggesting differences between various lupus - prone mouse models [ 23 , 24 , 61 , 96 ] .
administration of nh2-terminally truncated cx3cl1 analogs blocked cx3cl1/cx3cr1 interaction and significantly ameliorates glomerular and vascular lesions in mrl / lpr mice , reducing the infiltration of macrophages and cx3cr1 cells to the glomerular , interstitial , and perivascular regions .
t cells , however , were only reduced in the interstitial regions . with cx3cr1 blockade ,
the levels of circulating anti - dsdna igg and igg - containing ic were otherwise not affected , which , together with unchanged splenomegaly and lymphadenopathy , suggested a direct function of cx3cl1/cx3cr1 in the kidney that promotes ln progression in mrl / lpr mice . in sle patients ,
cx3cl1 expression is significantly increased in the glomeruli in class iv glomerulonephritis compared to other classes .
in addition , glomerular cx3cl1 expression is positively correlated with the infiltration of glomerular cd16 cells that express cx3cr1 , which deteriorates lupus disease , suggesting the clinical involvement of cx3cl1/cx3cr1 in ln development . aside from the t cell - related chemokine receptors discussed above , cxcr6 , ccr4 , and ccr6 that are associated with the recruitment of th1 , th2 , and th17/treg , respectively , have been also studied in sle [ 16 , 97 , 98 ] .
cxcr6 and its ligand cxcl16 have been shown to be involved in autoimmune diseases such as rheumatoid arthritis .
blockade of cxcl16 in mice also attenuates glomerulonephritis induced by antiglomerular basement membrane antibodies . in both mrl / lpr and nzb / w f1 mice , the expression of cxcr6 and cxcl16
while the lack of available blocking antibodies has hindered the investigation of the role of cxcr6/cxcl16 in ln , cxcr6 has been shown to facilitate the infiltration of activated cd8 t cells to the inflamed liver .
it is thus possible that cxcr6 cd8 t cells may be recruited into the inflamed kidney in ln through a cxcl16-dependent mechanism .
moreover , it has been shown that the level of soluble cxcl16 ( scxcl16 ) in the serum of sle patients is significantly higher compared to hc and is positively correlated with sle disease activity index ( sledai ) of patients .
in addition , the concentration of scxcl16 drops with disease remission . therefore , cxcl16 may be involved in ln development by recruiting cxcr6 t cells into the nephritic kidney .
ccr4 has two chemokine ligands , ccl17 ( thymus and activation - regulated chemokine , tarc ) and ccl22 ( macrophage - derived chemokine ) .
the expression of renal ccr4 and its two ligands is increased in mrl / lpr mice as ln progresses .
interestingly , blockade of ccl22 , but not ccl17 , in mrl / lpr mice led to reduced proteinuria and serum creatinine with improved renal function [ 56 , 103 ] .
moreover , the number of ccr4 t cells is reduced in the peripheral blood of sle patients compared to hc , suggesting increased migration of these cells into inflamed tissues .
accordingly , ccr4 cells are found in the kidney of sle patients that colocalize with cd4 cells .
thus , ccr4 t cells may selectively use ccl22 to migrate into the lupus nephritic kidney .
ccr6 is the chemokine receptor for ccl20 ( liver and activation - regulated chemokine , larc , or macrophage inflammatory protein 3 , mip-3 ) .
ccr6 is expressed on t cells , preferentially on th17 and treg cells [ 40 , 104110 ] . the interaction of ccr6 and ccl20 can recruit treg and th17 cells into the kidney in murine nephrotoxic nephritis [ 98 , 111 ] . whether this interaction can recruit th17 cells into the kidney to promote ln , or recruit treg cells to attenuate ln , remains to be explored . in nzb / w f1 mice , the expression of ccr6 and ccl20
these results suggest that ccr6 and ccl20 may function to regulate ln by recruiting th17 and treg cells .
as summarized above , in the kidney of both sle patients and lupus - prone mice , many chemokines rarely expressed at steady state are induced or significantly increased with ln progression , suggesting that local and/or systemic inflammatory factors may trigger the upregulation of these chemokines .
as summarized in table 3 , targeting both renal parenchymal cells and renal - infiltrating immune cells , nucleic acid - containing antigens and autoantibodies are considered to be the major inflammatory stimulators initiating and/or accelerating chemokine release in the lupus nephritic kidney .
mesangial cells and other intrinsic renal cells like glomerular capillary endothelial and proximal tubular epithelial cells that express several toll - like receptors ( tlrs ) have the potential to be activated by different antigens to produce inflammatory factors including chemokines .
polyi : c rna that mimics viral dsrna can induce mesangial cells from mrl / lpr mice to produce ccl2 , whereas mesangial cells from humans can be activated by polyi : c to produce cxcl1 through the tlr3 signaling pathway [ 112 , 113 ] .
mesangial cells from lupus - prone mice , compared to nonlupus mice , were more sensitive to lipopolysaccharide ( lps ) stimulation as shown by the higher tlr4 , myd88 , and nfb expression and higher ccl2 production , suggesting a mechanism of how bacterial infections accelerate lupus disease . besides exogenous factors , primary mesangial cells isolated from nzb / w f1 mice , upon self - nucleosome or nucleosome - containing ic stimulation in vitro , have been shown to produce several chemokines including ccl2 , ccl7 , ccl20 , cxcl2 , and cxcl5 , suggesting self - antigen and autoantibody - mediated mesangial activation . regarding autoantibody - induced mesangial activation
, it has been shown that pathogenic anti - dsdna igg can upregulate cxcl1 and cx3cl1 transcripts and the secretion of cxcl1 from mesangial cells isolated from mrl / lpr mice through both fc receptor- ( fcr- ) dependent and independent pathways .
further studies have shown that fcr - independent pathway is dependent on tlr2/4 and the receptor for advanced glycation end products ( rage ) but independent of dna / tlr9 , as pathogenic anti - dsdna igg clone 1a3f can bind high mobility group box 1 , an endogenous ligand for tlr2/4 and rage , through which 1a3f activates tlr2/rage - myd88-nfb pathway in mesangial cells , leading to the production of several chemokines including cxcl1 , cxcl2 , cxcl5 , cxcl16 , ccl7 , and ccl20 [ 96 , 116 ] .
autoantibodies can also indirectly activate intrinsic renal cells . when incubated with immobilized igg mimicking ic deposition in the kidney ,
lymphocytes isolated from human pbmc can be activated in a fcr - dependent way to produce il-1 that in turn stimulates human mesangial cells , glomerular capillary endothelial cells , and proximal tubular epithelial cells to further produce ccl2 . moreover , a transcription factor , fli1 , has been shown to directly bind the promoter region of ccl2 and ccl5 genes to promote their expression in primary endothelial cells of the kidney in nzm2410 lupus - prone mice [ 118 , 119 ] .
in addition to the activation of renal parenchymal cells , renal - infiltrating macrophages and dendritic cells have been shown to produce several chemokines upon stimulation by tlr2/4 , tlr3 , tlr7 , and tlr9 ligands .
biglycan , an endogenous stimulator of tlr2/4 , is increased in the serum and kidney of both sle patients and mrl / lpr mice .
an in vitro study further shows that macrophages and dendritic cells produce cxcl13 upon biglycan activation of tlr2/4-ros signaling pathway that is independent of inflammasome .
polyi : c rna mimicking viral dsrna and a tlr7 agonist mimicking viral ssrna can induce macrophages and dendritic cells isolated from mrl / lpr mice to produce ccl2 through the tlr3 and tlr7 signaling pathways , respectively [ 112 , 121 ] .
in addition , it has been shown that tlr7 and tlr9 are mostly detected in renal - infiltrating macrophages and dendritic cells rather than intrinsic renal cells of mrl / lpr mice [ 26 , 121 ] .
cpg , mimicking bacterial dna or self - dsdna , can induce ccl2 , ccl5 , and ccr5 in the kidney through the tlr9 pathway when injected into mrl / lpr mice .
detailed studies have shown that exogenous cpg or bacterial dna particularly bind to infiltrating macrophages and dendritic cells in the glomerular and tubulointerstitial regions of the kidney in mrl / lpr mice .
chloroquine - blocked tlr9 pathway abolishes ccl5 induction in spleen monocytes , further demonstrating tlr9-dependent chemokine induction in renal - infiltrating innate immune cells upon cpg - dna triggering .
finally , mirna-125a has been shown to indirectly downregulate ccl5 production by activated t cells through targeting kruppel - like factor 13 ( klf13 ) .
it has been demonstrated that mirna-125a is downregulated while klf13/ccl5 are upregulated in pbmc of sle patients compared to healthy controls , suggesting that dysregulation of ccl5 in sle patients is dependent on mirna-125a .
to date , renal biopsy is still the gold standard for accurate diagnosis and classification of ln and for the prognosis of ln activity and chronicity in patients upon treatment .
however , chemokines in the urine of lupus nephritic patients have been studied according to established diagnosis of ln classes that suggest their potential use as noninvasive biomarkers for ln activity monitoring , treatment responses , and remission / flare prediction after the biopsy diagnosis .
urine chemokines can be used to supplement renal biopsy diagnosis of ln . in both adult and juvenile sle patients , urinary ccl2 ( uccl2 )
concentration is significantly higher in nephritic patients than nonnephritic patients and healthy controls [ 123 , 124 ] .
moreover , both protein and mrna levels of uccl2 are significantly higher in sle patients with active ln compared to those with inactive ln [ 77 , 125130 ] .
further studies have shown that uccl2 alone or combined with other factors can distinguish different classes of ln , as uccl2 concentration is positively correlated to progressive ln classes and significantly increased in diffuse proliferative group compared to focal proliferative and mesangioproliferative groups [ 123 , 131 , 132 ] .
ln , it is important to monitor uccl2 whose level is high during interstitial inflammation in moderate - severe sle patients . besides uccl2 , urinary cxcl10 ( ucxcl10 ) concentration is also significantly higher in nephritic patients than nonnephritic sle patients . a cut - off value 93  pg / dl of ucxcl10 has been proved to be a good prediction of nephritis with high sensitivity and specificity .
also , ucxcl10 concentration is positively correlated with renal activity score and renal biopsy grade .
in addition , ucxcl10 and cxcr3 mrna levels from class iv nephritic patients are increased compared to other classes .
similarly , the urinary cxcl16 level can also distinguish inactive and active ln in sle patients . during the treatment of ln
uccl2 has been shown to be a good biomarker to predict juvenile ln improvement . in this study ,
the cut - off uccl2 concentration is 343  pg / ml , with a value lower than that predicting an improved renal disease activity .
two other studies have also shown that uccl2 is reduced in sle patients with complete or partial ln remission , while its level is maintained in nonremission patients , suggesting uccl2 as a good marker for prognosis [ 129 , 132 ] .
similarly , ucxcl10 is reduced in sle patients upon remission into inactive ln in a longitudinal follow - up study , suggesting ucxcl10 is also a good biomarker for monitoring ln improvement of sle patients following the treatment .
after the remission , the elevation of uccl2 can be detected 2 to 4 months prior to another ln flare , and changes of uccl2 concentration can distinguish different levels of ln flare severity , suggesting that uccl2 may be a good marker for predicting recurring ln flares [ 129 , 132 ] .
as chemokines and chemokine receptors are important in the recruitment of leukocytes to the kidney in the development of ln , one would naturally think of developing new treatments for ln that target the interaction between chemokines and chemokine receptors .
however , the design of such treatments should take into consideration the potential limitations ( discussed below ) , as many commercial drugs designed to target chemokines / chemokine receptors in different diseases have unfortunately failed in clinical trials ( summarized tables in references ) [ 138 , 139 ] .
the complexity of chemokine and chemokine receptor system and possible redundancy are a challenge for the development of new drugs to block leukocyte infiltration [ 139 , 140 ] .
some chemokines , such as ccl5 , can recognize several chemokine receptors ( ccr1 , ccr3 , and ccr5 ) , whereas some chemokine receptors , such as cxcr3 , can interact with different chemokines ( cxcl9 , cxcl10 , and cxcl11 ) .
current drugs including small chemical molecules and monoclonal antibodies are designed to simply block the interactions between chemokines and chemokine receptors by neutralizing either chemokines or chemokine receptors , which is insufficient to pinpoint the specific function of each chemokine / chemokine receptor pair .
thus , detailed studies on the dynamic interactions and functions of each particular chemokine / chemokine receptor pair in the specific diseases are critical for successful drug development .
in addition , the chemokine and chemokine receptor redundancy is reflected in a situation where one chemokine receptor may function in compensation of another if the other chemokine receptor is blocked .
leukocytes always express more than one type of chemokine receptors on the surface , so blocking the ligation of one chemokine receptor may not completely or efficiently prevent the infiltration of leukocytes . for example , while both ccr5 and cxcr3 have been shown to promote organ transplantation rejection by inducing t cells infiltration in the transplanted organ , their functions seem to be redundant .
ccr5 and cxcr3 double blocking compared to either single blocking makes a much greater prolonged allograft survival in a murine heterotopic heart transplantation model .
another challenge for ln drug development that involves chemokines and chemokine receptors is to achieve cell - specific targeting . to this end
, studies of chemokine receptor expression at the single cell level may help identify the cell type of interest .
for example , renal - infiltrating t cells have been shown to express ccr1 , ccr4 , ccr5 , cxcr3 , and cxcr5 [ 24 , 50 , 67 , 70 , 87 ] . however , it is unknown whether the chemokine receptors are expressed on the same t cell subsets or differentially expressed on distinct t cell subsets .
if we can define t cell subsets by using different combinations of chemokine receptors , we may be able to more specifically target pathogenic t cells by blocking the corresponding chemokine receptors .
an additional limitation is the use of lupus - prone mice where most of the mechanistic studies are performed to better understand the pathogenesis of ln .
the differences between human patients and mouse models make it difficult to translate the results of mouse studies to successful clinical trials .
therefore , it is important to study the differences and similarities between sle patients and lupus - prone mice regarding their use of chemokines and chemokine receptors .
an example is cxcl9 , which is preferentially used in mice but not in humans , versus cxcl10 , which appears to be the predominant chemokine in the kidney of sle patients [ 23 , 67 ] .
finally , how to specifically deliver chemokine - based drugs into the kidney is another important question .
it is difficult to find a kidney - specific chemokine or chemokine receptor critical for the development of ln .
systemically blocking a chemokine or chemokine receptor will likely lead to many outcomes other than attenuating ln , causing negative side effects such as an increased chance of infection or cancer .
therefore , while targeting the interaction between chemokines and chemokine receptors is a promising avenue , further studies are required to dissect and better understand the mechanisms behind such interactions before a chemokine - based drug can be developed to treat ln .
although many commercial chemokine receptor antagonists failed to reach expectations in treating different diseases , targeting chemokines / chemokine receptors may still be a promising strategy in ln .
first , studies using sle patient cells / tissue and animal models summarized in this review have demonstrated the involvement of chemokines / chemokine receptors in ln progression , suggesting the potential of targeting this system in ln treatment .
second , the failure of previously designed drugs is due to our insufficient understanding of the complicated chemokine / chemokine receptor system , which can be improved by further studies .
third , with better understanding of chemokine / chemokine receptor system , future drugs designed to more specifically target particular chemokine and chemokine receptor interactions will minimize the off - target effects and side effects commonly observed for immunosuppressive drugs and monoclonal antibodies , which are nonspecific .
finally , we may be able to learn from pathogens that are known to specifically target chemokine / chemokine receptor pairs   to design better drugs with improved specificity .
 lupus nephritis ( ln ) is a major cause of morbidity and mortality in the patients with systemic lupus erythematosus ( sle ) , an autoimmune disease with damage to multiple organs . 
 leukocyte recruitment into the inflamed kidney is a critical step to promote ln progression , and the chemokine / chemokine receptor system is necessary for leukocyte recruitment . in this review , 
 we summarize recent studies on the roles of chemokines and chemokine receptors in the development of ln and discuss the potential and hurdles of developing novel , chemokine - based drugs to treat ln . 