1Division of Nephrology, 2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Submitted 19 December 2002 ; accepted in final form 20 April 2003
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ABSTRACT |
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Th1/Th2 cells; inflammation; interleukin-4
CD4 T cells functionally differentiate into two different phenotypes, Th1
and Th2 cells (1).
Differentiation into Th1 and Th2 cells is characterized by specific cytokine
expression. IL-12 is required for Th1 cell differentiation, and this is
followed by the production of IFN-, whereas Th2 cells require IL-4
production followed by IL-4, IL-5, IL-6, IL-10, and IL-13 secretion
(1,
27). Given recent data
supporting a role for T cells, particularly the CD4+ T cell in
renal IRI, we hypothesized that the Th1 inflammatory pattern is pathogenic in
renal IRI, whereas the Th2 pattern is protective (opposite to what has been
proposed in asthma) (26).
Recent availability of gene knockout mice for signal transducers and
activators of transcription (STAT) 4, which mediate Th1 differentiation, or
STAT6, which is required for Th2 differentiation, allowed us to investigate
this question (9,
10). We evaluated mice
deficient in STAT4 and STAT6 and compared their cytokine responses and
outcomes in experimental renal IRI. We found that the T cells from
STAT4/ mice had a blunted IFN-
response to stimulation,
and those from STAT6/ mice had diminished IL-4. The
STAT6/ mice had a marked worsening of kidney injury following
renal IRI, with worse function and structural injury. In contrast,
STAT4/ mice had modest and limited protection from renal IRI. To
evaluate if blunted IL-4 production was the mechanism underlying worse renal
IRI in STAT6/, IL-4/ mice were studied.
IL-4/ mice demonstrated a marked worsening of postischemic
phenotype similar to STAT6/ mice, suggesting that the IL-4
deficiency in the STAT6/ was a likely mechanism conferring worse
injury. Inflammatory pathways may serve protective or deleterious functions in
renal IRI.
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METHODS |
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Cell characterization. Intracellular cytokine staining (ICCS) was
used to characterize the cells obtained from STAT-deficient mice. An aliquot
of T cell suspension from spleen was stimulated with anti-CD3 antibody (CD3
chain, 1452C11, BD Pharmingen, San Diego, CA) in the presence of
Brefeldin A (GolgiPlug, BD Pharmingen) in RPMI containing 10% fetal bovine
serum. After 12 h of stimulation, cells were harvested, treated with
anti-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 specific
to an intracellular cytokine, such as IFN-
(XMG1.2, BD Pharmingen) and
IL-4 (11B11, BD Pharmingen), under continuous saponin permeabilization
(Perm/Wash, BD Pharmingen), and then analyzed.
Renal IRI. Mice were anesthetized using a ketamine-xylazine
mixture (150 µg/g ketamine, 3 µg/g xylazine), and an incision was made
on the central abdomen. Avoiding intestines and bowel, microvascular clamps
were applied to bilateral renal pedicles. After 35 min of renal ischemia,
clamps were removed and the incision was closed. During the procedure, mice
were 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 serum creatinine as a direct measure of kidney function up to 72 h postischemia (16). At 0, 24, 48, and 72 h postischemia, 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 tubular injury. The magnitude of cell loss and necrotic codes were scored based on five levels. Scores ranged from zero to four based on the percentage of tubules affected (0: <10%; 1: 10 to 25%; 2: 25 to 50%; 3: 50 to 75%; 4: >75%).
Frozen kidney sections at baseline, 24, and 72 h postischemia were stained with a monoclonal antibody specific to neutrophils (Ly-6G, Gr-1, BD Pharmingen). Stained cells in the corticomedullary zone were counted in a blinded fashion (10 random field were counted at x200 magnification).
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) in ice-cold KPO4 buffer. Samples were spun at 17,000 g for 30 min at 4°C, and pellets were washed and spun an additional two times. Then 0.5% hexadecyltrimethylammonium bromide-10 mM EDTA was added to the remaining pellet (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 was recorded at 460 nm. One unit of MPO was defined as a change of absorbance of one per minute. Results were expressed as percent baseline of units MPO per gram of protein that was detected using a bicinchoninic assay (Pierce Chemical, Rockport, IL).
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 was
evaluated by the presence of GAPDH housekeeping RNA and analyzed as a percent
increase from "0" hour samples. A ribonuclease protection assay
(RPA) was used to characterize the proinflammatory gene expression. We used
the mouse-specific multiple cytokine assays RIBOQUANT from Pharmingen. Focus
was on the following cytokines: IL-1, TNF-
, ICAM-1, and IL-6
based on previous studies implicating them as potential mediators of renal IRI
(13).
Statistics. One-way ANOVA was used for the comparison among the group of data, using SigmaStat software (SPSS Science, Chicago, IL). Data are expressed as means ± SE. A P value <5% was considered to be significant.
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RESULTS |
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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 at 24, 48, and 72 h postischemia (P < 0.05). STAT4/ mice, however, were only modestly protected from renal IRI compared with Balb/c wild type at 24 h postischemia and not at other time points (Fig. 2).
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Tubular injury scoring of STAT4- and STAT6-deficient mice. H&E-stained kidney tissues postischemia were scored to determine the severity of tubular injury. STAT6/ mice had significantly higher tubular injury scores compared with wild-type mice. Tubular injury in STAT4/ mice was significantly better than STAT6/ mice but comparable to wild type (Fig. 3).
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Polymorphonuclear cell counts. Polymorphonuclear cell counts were evaluated by specific staining to determine whether enhanced infiltration in STAT6/ mice was a potential mechanism of worse injury postischemia. Although PMN counts increased postischemia, this was similar in both STAT-deficient groups and wild-type mice at both 24 and 72 h postischemia (Fig. 4A).
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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 worse injury by augmenting phagocyte (macrophage and neutrophil) infiltration postischemia. MPO levels also increased postischemia but similarly in STAT-deficient and wild-type mice (Fig. 4B).
Proinflammatory gene expression. IL-1, TNF-
, ICAM-1,
and IL-6 have been implicated as mediators of renal IRI
(Fig. 5). As previously
described (13), gene
expression of these molecules measured by RPA increased from baseline
postischemia; however, there was no significant difference in the regulation
of these genes between the STAT-deficient and wild-type mice.
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IL-4- and IFN--deficient mice. In view of the
significant reduction in IL-4 production by T cells in the
STAT6/ mice, we hypothesized that IL-4 deficiency was a
potential mechanism by which the STAT6/ mice had the worsened
postischemic response. IL-4-deficient mice also demonstrated a markedly worse
functional and histological response after IRI (Figs.
6 and
7). IFN-
-deficient mice
were studied because of the deficiency in IFN-
in STAT4/
mice. The IFN-
-deficient mice had similar degrees of tubular injury and
renal dysfunction postischemia as wild-type mice.
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DISCUSSION |
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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, also
support a pathogenic role of T cells
(8,
14,
19,
32). However, the mechanisms
underlying these effects have not been elucidated
(17). One approach in the
exploration of the T cell involvement in renal IRI is to examine whether the T
cell involvement is via the CD4 cell polarization model into Th1 and Th2
cytokine-producing cells. Proinflammatory cytokines involved in T cell
polarization, such as IFN-, IL-6, and IL-10, have been reported to be
upregulated in postischemic kidneys from wild-type animals
(6,
13). When CD4
"helper" T cells functionally differentiate into Th1 and Th2
cells, they require the specific STAT proteins, STAT4 and STAT6. The STAT4
pathway is activated by the conjugation of IL-12 and IL-12R on Th1 cells
followed by IFN-
production. The STAT6 pathway is activated by the
conjugation 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 and
inhibits Th2 effector cell functions, whereas IL-4 promotes Th2 functions and
inhibits 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 would be deleterious in renal IRI and Th2 protective, we originally compared the responses to renal IRI in Balb/c mice compared with C57BL/6. This is based on their relative tendencies to express the Th2 and Th1 phenotypes, respectively (7). Although we found a mild susceptibility to IRI in C57BL/6 mice, these data only suggested the possibility of this paradigm in IRI (3). With the availability of specific knockouts of the key T cell cytokine-polarizing STAT4 and STAT6 genes, this question could now be addressed more directly.
We initially confirmed that the STAT4 mice that we used had a deficiency in
CD4 T cell polarization into Th1 cells using ICCS of T cells. The marked
impairment in IFN- and increased IL-4 demonstrated this. We then
evaluated STAT6-deficient CD4+ cells and found the inverse T cell
phenotype. Proceeding with renal IRI studies, we found a very modest but
reproducible functional protection from renal IRI in the STAT4/
mice, although histological protection was not seen. We interpreted the mild
functional protection in STAT4/ as likely minor and was not
sufficient to be reflected in the visual tubular injury assessment.
Alternatively, we cannot exclude a mild "functional" effect on
renal blood flow. We had been focusing on identifying deleterious pathways in
renal IRI and did not expect the unusually severe renal injury phenotype in
the STAT6/ mice, suggesting that this pathway is protective in
renal IRI. The markedly increased functional injury in the
STAT6/ mice corresponded to their enhanced tubular injury
postischemia, and we therefore turned our focus to this potentially protective
pathway.
We then explored potential effector mechanisms by which the
STAT6/ mice had enhanced injury. In view of previous data
implicating the neutrophil as being a participant in postischemic renal injury
(12), we measured neutrophil
influx into postischemic kidney in the wild-type and STAT-deficient mice using
specific monoclonal staining. We did not observe a significant difference in
the postischemic neutrophil influx between groups. We also used the
alternative technique of renal MPO measurement, which primarily detects
infiltrating macrophages, but also neutrophils in a more observer-independent
fashion than cell counting
(31). However, although MPO
levels increased in all groups postischemia, they did not correlate with the
significantly 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 technique
that 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 these
genes increased postischemia, their increase did not correlate with the
changes in the STAT6/ mice.
A major characteristic phenotype of the STAT6/ mouse is the
deficiency of T cell production of IL-4. We therefore hypothesized that IL-4
deficiency could be a major mechanism by which the STAT6/ mice
had worse injury after renal IRI. We therefore studied IL-4-deficient mice,
which were subsequently found to have a marked worsening of renal function and
structure postischemia. The finding that the major "Th2" cytokine
IL-4 is likely a protective factor in renal IRI is consistent with the finding
by 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/ mouse are consistent with work in the liver using a different approach: IL-4, administered to promote the STAT6 gene, resulted in protection from postischemic liver injury (11, 30). The contribution of the STAT4 gene to liver IRI was also found to be more subtle, with liver IRI in STAT4/ mice revealing the protection only under the circumstance of endogenous IL-12 blockade (11). The lesser role of STAT4 compared with STAT6 in liver IRI is consistent with our current findings in the kidney. Recently, the STAT6 and STAT4 injury paradigm in liver IRI has been linked to heme oxygenase production (20). It is also important to note that although IL-4 is a prototypic Th2 CD4 cell-secreted cytokine, IL-4 can also be made by NK cells, mast cells, basophils, and undifferentiated T cells (15). In preliminary studies to identify the cell source of IL-4 in ischemia using T cell-adoptive transfer methods, it appeared that the T cell might be a partial source (data not shown).
The identification of the protective role of the STAT6 pathway in renal IRI unveils a novel area of investigation in ARF. In addition, these data suggest the existence of a protective Th2 paradigm in ischemic renal injury. Modification of this signaling pathway and modifying IL-4 responses could lead to new therapeutics for IRI.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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