Contrasting roles for STAT4 and STAT6 signal transduction pathways in murine ren



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作者：Naoko Yokota, Melissa Burne-Taney, Lorraine Racusen, and Hamid Rabb作者单位：1 Division of Nephrology, 2 Department of Pathology, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland 21205
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      【摘要】% Z3 O1 V; [* B" P
   Recent data support a modulatory role for CD4 T cells in experimental renalischemia-reperfusion injury (IRI). CD4 T cells can functionally differentiateto either a Th1 (IFN- producing) or the counterbalancing Th2 (IL-4)phenotype. The enzymes signal transducers and activators of transcription(STAT) 4 and STAT6 regulate Th1 or Th2 differentiation and cytokineproduction, respectively. We therefore hypothesized that mice that were STAT4deficient would be protected from renal IRI and that STAT6-deficient micewould have a more severe course. Intracellular cytokine staining ofsplenocytes from STAT4-/- or STAT6-/- exhibiteddistinct IFN- and IL-4 cytokine expression profiles.STAT6-/- had markedly worse renal function and tubular injurypostischemia compared with wild type. STAT4-/- had only mildly improved function. Renal phagocyte infiltration and ICAM-1 upregulation weresimilar in STAT4-/-, STAT6-/-, and wild type. Toevaluate if the mechanism of the marked worsening in the STAT6-/-mice could be due to IL-4 deficiency, IL-4-deficient mice were studied and hadsimilar postischemic phenotype to STAT6-/- mice. These datademonstrate that the STAT6 pathway has a major protective role in renal IRI.IL-4 deficiency is a likely mechanism underlying the STAT6 effect. A"yin-yang" role for inflammation is emerging in renal IRI, similarto recent observations in atherosclerosis.
      【关键词】 Th/Th cells inflammation interleukin
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ISCHEMIA - REPERFUSION INJURY (IRI) is the main cause of intrinsic acute renal failure (ARF). Native kidney ARF is associated with a greater than30% mortality ( 22, 24 ), and transplant ischemic ARF adversely affects both short- and long-term allograft function ( 25 ). Inflammation iscurrently established to be an important pathogenic component in thedevelopment of renal IRI, but the cellular basis is incompletely understood.Although the neutrophil was originally focused on as the major inflammatorymediator of renal IRI, recent data support an important role for the T cell.Mononuclear leukocytes are found in the vasa recta in biopsies of patientswith ischemic ARF ( 21 ).CTLA-4Ig, which blocks T cell costimulation via the CD28-B7 pathway, improved cold renal IRI in a rat model( 23 ). Furthermore, treatment with a monoclonal antibody specific to B7 molecule has also been found to beprotective in a rat model of renal IRI( 4 ). Targeted gene knockoutmice have enabled more detailed mechanistic evaluation of T cell pathways indisease. CD4 and CD8 double-knockout mice are significantly protected fromrenal IRI ( 18 ). Nu/nu mice, which are T cell deficient, are also protected from renal injurypostischemia and the injury phenotype is restored after adoptive transfer ofnaive T cells into nu/nu mice( 2 ). Furthermore, CD4 knockoutmice are also significantly protected from renal IRI, whereas CD8 knockoutmice are not. Adoptive transfer of naive CD4 T cells into CD4 knockout miceresulted in reconstitution of the injury level( 2 ). IFN- and CD28pathways were shown to play an important role in T cell-mediated injury inthis study. Mice depleted of T cells, particularly CD4 cells, using monoclonalantibodies, are also afforded significant protection from renal dysfunctionpostischemia ( 28 ).
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CD4 T cells functionally differentiate into two different phenotypes, Th1and Th2 cells ( 1 ).Differentiation into Th1 and Th2 cells is characterized by specific cytokineexpression. IL-12 is required for Th1 cell differentiation, and this isfollowed by the production of IFN-, whereas Th2 cells require IL-4production followed by IL-4, IL-5, IL-6, IL-10, and IL-13 secretion( 1, 27 ). Given recent datasupporting a role for T cells, particularly the CD4 T cell inrenal IRI, we hypothesized that the Th1 inflammatory pattern is pathogenic inrenal IRI, whereas the Th2 pattern is protective (opposite to what has beenproposed in asthma) ( 26 ).Recent availability of gene knockout mice for signal transducers andactivators of transcription (STAT) 4, which mediate Th1 differentiation, orSTAT6, which is required for Th2 differentiation, allowed us to investigatethis question ( 9, 10 ). We evaluated micedeficient in STAT4 and STAT6 and compared their cytokine responses andoutcomes in experimental renal IRI. We found that the T cells fromSTAT4-/- mice had a blunted IFN- response to stimulation,and those from STAT6-/- mice had diminished IL-4. TheSTAT6-/- mice had a marked worsening of kidney injury followingrenal IRI, with worse function and structural injury. In contrast, STAT4-/- mice had modest and limited protection from renal IRI. Toevaluate if blunted IL-4 production was the mechanism underlying worse renalIRI in STAT6-/-, IL-4-/- mice were studied.IL-4-/- mice demonstrated a marked worsening of postischemicphenotype similar to STAT6-/- mice, suggesting that the IL-4 deficiency in the STAT6-/- was a likely mechanism conferring worseinjury. Inflammatory pathways may serve protective or deleterious functions inrenal IRI.: c" P% E. W, ]9 q

METHODS
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Mice. STAT4-/-(C.129S2-Stat4 tm1Gru ), STAT6-/- (C.129S2-Stat6 tm1Gru ), IL-4-/-(Balb/c-IL-4 tm2Nnt ), and IFN- -/-[C.129S7(B6)-Ifng tm1Ts ] were purchased from JacksonLaboratory (Bar Harbor, ME). Background strain-, age-, and sex-matched controlmice were used. Mice were held under pathogen-free conditions according toNational Institutes of Health guidelines.
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Cell characterization. Intracellular cytokine staining (ICCS) wasused to characterize the cells obtained from STAT-deficient mice. An aliquotof T cell suspension from spleen was stimulated with anti-CD3 antibody (CD3 chain, 145-2C11, BD Pharmingen, San Diego, CA) in the presence ofBrefeldin A (GolgiPlug, BD Pharmingen) in RPMI containing 10% fetal bovineserum. After 12 h of stimulation, cells were harvested, treated withanti-mouse CD16/CD32, and stained with pan-T cell surface marker CD90.2 (Thy-1.2, 30-H12, BD Pharmingen). Cells were fixed with 1% formaldehyde (Cytofix/Cytoperm, BD Pharmingen) and stained with a second antibody specificto an intracellular cytokine, such as IFN- (XMG1.2, BD Pharmingen) andIL-4 (11B11, BD Pharmingen), under continuous saponin permeabilization(Perm/Wash, BD Pharmingen), and then analyzed.' F2 J7 c7 H. S2 {& [# D- ]6 K& K
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Renal IRI. Mice were anesthetized using a ketamine-xylazine mixture (150 µg/g ketamine, 3 µg/g xylazine), and an incision was madeon the central abdomen. Avoiding intestines and bowel, microvascular clampswere applied to bilateral renal pedicles. After 35 min of renal ischemia,clamps were removed and the incision was closed. During the procedure, micewere well hydrated and their body temperature was controlled at 37°C using an adjustable heating pad. Animals were kept under veterinarian observation postischemia.

Analysis of renal function. Serum creatinine was used for the evaluation of renal function postischemia. Previous work validated serumcreatinine as a direct measure of kidney function up to 72 h postischemia( 16 ). At 0, 24, 48, and 72 hpostischemia, mice were bled from their tail vein using hematocrit tubes. Serum samples were analyzed on a COBAS Mira chemical analyzer (Roche, Basel,Switzerland), using creatinine reagent 557 (Sigma, St. Louis, MO).

Histological and immunohistochemical analysis. Formaldehyde-fixed paraffin sections of kidney were stained with hematoxylin and eosin (H&E).A "blinded" renal histologist scored the degrees of tubularinjury. The magnitude of cell loss and necrotic codes were scored based onfive levels. Scores ranged from zero to four based on the percentage oftubules affected (0: 75%).
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Frozen kidney sections at baseline, 24, and 72 h postischemia were stainedwith a monoclonal antibody specific to neutrophils (Ly-6G, Gr-1, BDPharmingen). Stained cells in the corticomedullary zone were counted in ablinded fashion (10 random field were counted at x 200magnification)., m, r' a: W1 n# W; c; g2 W  F

Leukocyte detection using myeloperoxidase assay. Myeloperoxidase assay (MPO) levels were used to detect phagocytes (neutrophil and macrophage)in mouse kidney. Briefly, kidney samples were homogenized (1:20 wt/vol) inice-cold KPO 4 buffer. Samples were spun at 17,000 g for 30min at 4°C, and pellets were washed and spun an additional two times. Then0.5% hexadecyltrimethylammonium bromide-10 mM EDTA was added to the remainingpellet (6:1). Suspensions were sonicated and freeze-thawed three times, then incubated at 4°C for 20 min. After final centrifugation at 17,000 g at 4°C for 15 min and addition of assay buffer (4:1),supernatants were measured for MPO. Change in absorbance over 3.5 min wasrecorded at 460 nm. One unit of MPO was defined as a change of absorbance ofone per minute. Results were expressed as percent baseline of units MPO pergram of protein that was detected using a bicinchoninic assay (PierceChemical, Rockport, IL).
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Renal mRNA cytokine expression. Cellular RNA was isolated from snap-frozen kidneys at 0, 24, and 72 h postischemia using TRIZOL Reagent(Invitrogen Life Technologies, Carlsbad, CA). The quantity of RNA wasevaluated by the presence of GAPDH housekeeping RNA and analyzed as a percentincrease from "0" hour samples. A ribonuclease protection assay(RPA) was used to characterize the proinflammatory gene expression. We usedthe mouse-specific multiple cytokine assays RIBOQUANT from Pharmingen. Focuswas on the following cytokines: IL-1, TNF-, ICAM-1, and IL-6based on previous studies implicating them as potential mediators of renal IRI( 13 ).
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Statistics. One-way ANOVA was used for the comparison among thegroup of data, using SigmaStat software (SPSS Science, Chicago, IL). Data areexpressed as means ± SE. A P value / ~$ x9 [8 v8 u$ w' H
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RESULTS3 T. Z/ c" m2 u+ n; f' N
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Functional characteristics of T cells fromSTAT4 - / - and STAT6 - / - mice. ICCS was performed to phenotype mice that were used in eachexperimental group. T cells from STAT4-/- mice expressed reducedIFN- and increased IL-4, whereas those from STAT6-/- miceindeed expressed increased IFN- and reduced IL-4( Fig. 1 ).
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Fig. 1. Cytokine characteristics of signal transducers and activators oftranscription (STAT)-deficient animals. T cells from STAT4-/- micedemonstrated a reduced production of IFN- and vigorous production ofIL-4 production. In contrast, T cells from STAT6-/- mice had anopposite production of cytokine pattern with active IFN- production andminimum IL-4 production.  ?- D# S' Z# D& x/ Z% E: `
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Renal function postischemia of STAT4 - / - and STAT6 - / - mice. STAT6-/-mice had markedly higher serum creatinine compared with Balb/c wild type at24, 48, and 72 h postischemia ( P . S/ @6 {0 [/ N* R5 S& L7 d
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Fig. 2. Serum creatinine postischemia in STAT-deficient mice. STAT6-/-mice had significantly worse renal function compared with wild-type Balb/c(and STAT4-/-) at 24, 48, and 72 h postischemia.STAT4-/- mice were modestly protected compared with Balb/c andonly at 24 h postischemia. * P

Tubular injury scoring of STAT4- and STAT6-deficient mice. H&E-stained kidney tissues postischemia were scored to determine theseverity of tubular injury. STAT6-/- mice had significantly highertubular injury scores compared with wild-type mice. Tubular injury inSTAT4-/- mice was significantly better than STAT6-/-mice but comparable to wild type ( Fig.3 ).
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Fig. 3. Tubular injury score. Hematoxylin and eosin (H&E)-stained kidneytissues at 72 h postischemia were assessed for levels of tubular injury by a"blinded" reviewer. STAT6-/- mice displayed much moresevere injury compared with wild-type and STAT4-/- mice.* P * E* B$ a5 ?, F# C2 \
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Polymorphonuclear cell counts. Polymorphonuclear cell counts wereevaluated by specific staining to determine whether enhanced infiltration inSTAT6-/- mice was a potential mechanism of worse injurypostischemia. Although PMN counts increased postischemia, this was similar inboth STAT-deficient groups and wild-type mice at both 24 and 72 h postischemia( Fig. 4 A ).7 m3 Z$ e! `+ J: r
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Fig. 4. Evaluation of polymorphonuclear (PMN) cells and macrophages in the kidneypostischemia. A : numbers of PMNs in outer medulla were counted inblinded fashion for tissues 24 and 72 h postischemia. Data were expressed asPMN counts/10 random high-power fields. There was a paucity of PMNs atbaseline, then counts increased postischemia, with no significant differencesfound among groups ( n = 12). B : MPO levels in the kidneywere analyzed for tissues 0, 24, and 72 h postischemia. MPO levels arereflective of macrophage and neutrophil counts. MPO levels are expressedcompared with control "0" time mice, arbitrarily called"100%," and subsequent times are shown as a percentage of thisbaseline. MPO levels increased postischemia; however, no significantdifferences were found among groups, similar to PMN counts ( n = 3).Open bars, Balb/c wild type; hatched bars, STAT4-/-; filled bars,STAT6-/-.( I' j+ ]5 a" a+ y2 B* E% G: R7 [
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MPO level. MPO levels in kidney tissue were measured as an alternative approach to assess whether the STAT6 deficiency led to worseinjury by augmenting phagocyte (macrophage and neutrophil) infiltrationpostischemia. MPO levels also increased postischemia but similarly inSTAT-deficient and wild-type mice ( Fig.4 B ).
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Proinflammatory gene expression. IL-1, TNF-, ICAM-1,and IL-6 have been implicated as mediators of renal IRI( Fig. 5 ). As previouslydescribed ( 13 ), geneexpression of these molecules measured by RPA increased from baselinepostischemia; however, there was no significant difference in the regulationof these genes between the STAT-deficient and wild-type mice.
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Fig. 5. Cytokine mRNA expression in the kidney postischemia. Ribonucleaseprotection assay was performed on kidney tissues preischemia, 24 and 72 hpostischemia. The increasing levels of IL-1, TNF-, and IL-6postischemia occurred similarly in wild-type as well as STAT4-/-and STAT6-/- postischemia ( n = 3; open bars, Balb/c wildtype; hatched bars, STAT4-/-; filled bars,STAT6-/-).( [, w9 b' [, k  V0 y2 c, M; W
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IL-4- and IFN- -deficient mice. In view of thesignificant reduction in IL-4 production by T cells in theSTAT6-/- mice, we hypothesized that IL-4 deficiency was apotential mechanism by which the STAT6-/- mice had the worsenedpostischemic response. IL-4-deficient mice also demonstrated a markedly worse functional and histological response after IRI (Figs. 6 and 7 ). IFN- -deficient micewere studied because of the deficiency in IFN- in STAT4-/-mice. The IFN- -deficient mice had similar degrees of tubular injury andrenal dysfunction postischemia as wild-type mice.
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Fig. 6. Serum creatinine postischemia in IL-4- or IFN- -deficient mice.IL-4-deficient mice had significantly worse renal function compared withstrain-matched wild-type controls at 24 h postischemia. IFN- -deficientmice were similar to wild-type postischemia. * P

Fig. 7. Tubular injury score for IL-4- and IFN- -deficient mice postischemia.H&E-stained kidney tissues at 72 h postischemia were scored for the levelof tubular injury in a blinded fashion. IL-4-/- mice displayedsignificantly more severe injury compared with wild-type andIFN- -/- mice, which were comparable. * P ) Q! u  M$ V$ ^' s, J6 B4 d
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DISCUSSION* W1 B0 I' J  T

These data demonstrate a major protective role of the STAT6 pathway inrenal IRI. Given the deficiency of IL-4 in the STAT6-/- mouse, andsimilar responses to renal IRI between STAT6-/- mice andIL-4-/- mice, IL-4 deficiency is a likely mechanism to explain theobservations in the STAT6 knockout. Renal neutrophil and macrophageinfiltration, as well as upregulation of proinflammatory genes such as ICAM-1,are unlikely to underlie the worse renal outcome in the STAT6-/-mice. In contrast, STAT4-/- conferred limited protection from IRI,restricted to modest early functional improvement. IFN- deficiency inSTAT4-/- was mimicked in the IFN- -/- mice, withsimilar early outcomes to wild type after renal IRI.6 V9 J. M) n7 M0 t
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A number of groups recently demonstrated a role for T cells in renal IRI( 2, 4, 18, 23, 28 ). Studies in several models of IRI in other organs, such as the liver, intestine, lung, and brain, alsosupport a pathogenic role of T cells( 8, 14, 19, 32 ). However, the mechanismsunderlying these effects have not been elucidated( 17 ). One approach in theexploration of the T cell involvement in renal IRI is to examine whether the Tcell involvement is via the CD4 cell polarization model into Th1 and Th2cytokine-producing cells. Proinflammatory cytokines involved in T cellpolarization, such as IFN-, IL-6, and IL-10, have been reported to beupregulated in postischemic kidneys from wild-type animals( 6, 13 ). When CD4"helper" T cells functionally differentiate into Th1 and Th2cells, they require the specific STAT proteins, STAT4 and STAT6. The STAT4pathway is activated by the conjugation of IL-12 and IL-12R on Th1 cellsfollowed by IFN- production. The STAT6 pathway is activated by theconjugation of IL-4 and IL-4R on Th2 cells followed by production of IL-4,IL-5, IL-6, IL-10, and IL-13. IFN- promotes Th1 effector functions andinhibits Th2 effector cell functions, whereas IL-4 promotes Th2 functions andinhibits Th1 functions. In a variety of disease models, such as asthma,leprosy, and transplant rejection, T cells are skewed into Th1 or Th2 pathways( 1, 27 ).

On the basis of the hypothesis that the Th1 immune response pattern wouldbe deleterious in renal IRI and Th2 protective, we originally compared theresponses to renal IRI in Balb/c mice compared with C57BL/6. This is based ontheir relative tendencies to express the Th2 and Th1 phenotypes, respectively ( 7 ). Although we found a mildsusceptibility to IRI in C57BL/6 mice, these data only suggested thepossibility of this paradigm in IRI( 3 ). With the availability ofspecific knockouts of the key T cell cytokine-polarizing STAT4 and STAT6genes, this question could now be addressed more directly.

We initially confirmed that the STAT4 mice that we used had a deficiency inCD4 T cell polarization into Th1 cells using ICCS of T cells. The markedimpairment in IFN- and increased IL-4 demonstrated this. We thenevaluated STAT6-deficient CD4 cells and found the inverse T cellphenotype. Proceeding with renal IRI studies, we found a very modest butreproducible functional protection from renal IRI in the STAT4-/- mice, although histological protection was not seen. We interpreted the mildfunctional protection in STAT4-/- as likely minor and was notsufficient to be reflected in the visual tubular injury assessment.Alternatively, we cannot exclude a mild "functional" effect onrenal blood flow. We had been focusing on identifying deleterious pathways inrenal IRI and did not expect the unusually severe renal injury phenotype in the STAT6-/- mice, suggesting that this pathway is protective inrenal IRI. The markedly increased functional injury in theSTAT6-/- mice corresponded to their enhanced tubular injurypostischemia, and we therefore turned our focus to this potentially protectivepathway.

We then explored potential effector mechanisms by which the STAT6-/- mice had enhanced injury. In view of previous dataimplicating the neutrophil as being a participant in postischemic renal injury( 12 ), we measured neutrophilinflux into postischemic kidney in the wild-type and STAT-deficient mice usingspecific monoclonal staining. We did not observe a significant difference inthe postischemic neutrophil influx between groups. We also used thealternative technique of renal MPO measurement, which primarily detectsinfiltrating macrophages, but also neutrophils in a more observer-independentfashion than cell counting ( 31 ). However, although MPOlevels increased in all groups postischemia, they did not correlate with thesignificantly worse renal function in the STAT6-/- mice. We then measured gene expression of IL-1, TNF-, ICAM-1, and IL-6, which have all been implicated as mediators of renal IRI. We used an RPA techniquethat is established to detect increases in these genes in the current model( 13 ) as well as associate protective changes with an intervention( 29 ). Again, although thesegenes increased postischemia, their increase did not correlate with thechanges in the STAT6-/- mice.! Q" S& C' X' y% I1 W1 ]
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A major characteristic phenotype of the STAT6-/- mouse is thedeficiency of T cell production of IL-4. We therefore hypothesized that IL-4deficiency could be a major mechanism by which the STAT6-/- micehad worse injury after renal IRI. We therefore studied IL-4-deficient mice,which were subsequently found to have a marked worsening of renal function andstructure postischemia. The finding that the major "Th2" cytokineIL-4 is likely a protective factor in renal IRI is consistent with the findingby Star et al. ( 5 ) that another"Th2" cytokine, IL-10, is also protective in renal IRI.IFN- deficiency, however, did not confer a protective phenotype.

Our findings of the enhanced renal injury in the STAT6-/- mouseare consistent with work in the liver using a different approach: IL-4,administered to promote the STAT6 gene, resulted in protection frompostischemic liver injury ( 11, 30 ). The contribution of theSTAT4 gene to liver IRI was also found to be more subtle, with liver IRI inSTAT4-/- mice revealing the protection only under the circumstanceof endogenous IL-12 blockade( 11 ). The lesser role of STAT4compared with STAT6 in liver IRI is consistent with our current findings in the kidney. Recently, the STAT6 and STAT4 injury paradigm in liver IRI hasbeen linked to heme oxygenase production( 20 ). It is also important tonote that although IL-4 is a prototypic Th2 CD4 cell-secreted cytokine, IL-4can also be made by NK cells, mast cells, basophils, and undifferentiated Tcells ( 15 ). In preliminarystudies to identify the cell source of IL-4 in ischemia using T cell-adoptivetransfer methods, it appeared that the T cell might be a partial source (datanot shown).7 }6 M! u8 v" W9 K$ B& {& p: V

The identification of the protective role of the STAT6 pathway in renal IRIunveils a novel area of investigation in ARF. In addition, these data suggestthe existence of a protective Th2 paradigm in ischemic renal injury.Modification of this signaling pathway and modifying IL-4 responses could leadto new therapeutics for IRI.% O6 D) x1 ]" P
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DISCLOSURES$ k2 D+ D$ x& O: R4 ~% l
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M. Burne-Taney was supported by a National Kidney Foundation ResearchFellowship award. H. Rabb was supported by a National Institutes of HealthGrant R0-1, National Kidney Foundation Clinical Scientist Award, andRoche Organ Transplant Foundation Award.9 @# M3 \2 d2 S2 U* d4 p0 B8 }
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