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NKT Cell Activation Mediates Neutrophil IFN- Production and Renal Ischemia-Reperfusion Injury¹

Li Li^*, Liping Huang^*, Sun-sang J. Sung^*, Peter I. Lobo^*, Michael G. Brown^*,,, Randal K. Gregg, Victor H. Engelhard^, and Mark D. Okusa^2,*,,

^* Department of Medicine, Department of Microbiology, The Carter Immunology Center, and Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908

	Abstract

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Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4⁺ T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4⁺ cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4⁺ T cells expressed the activation marker, CD69⁺, and adhesion molecule, LFA-1^high. Three hours after reperfusion, kidney IFN--producing cells were comprised largely of GR-1⁺CD11b⁺ neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (J18^–/–), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN--producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Acute kidney injury remains a significant health concern as the incidence continues to rise and mortality remains unacceptably high (1). Inflammation is an important early event in the cascade of intracellular responses that ultimately result in apoptosis and/or necrosis (2). A number of studies have identified inflammatory cells that infiltrate the kidney following ischemia-reperfusion (IR)³ injury (IRI) including T and B cells, macrophages, and neutrophils (3). Recent studies using T, NKT, and B cell-deficient mice and adoptive transfer methods have provided strong evidence for the role of CD4⁺ T cells in the pathogenesis of IRI (4, 5). Renal tissue injury was markedly reduced in RAG-1^–/– mice, which are deficient in T, B, and NKT cells, when subjected to renal IRI (5). This protective effect was reversed following adoptive transfer of CD4⁺ T cells. These results suggest that CD4⁺ T cells contribute importantly to the early events in IRI. Because CD4⁺ T cells consist of functionally distinct subsets of T cells including Th cells and NKT cells, it became important to determine whether both subsets of T cells participate in early IRI, which primarily involves innate mechanisms. CD4⁺ T cells, including NKT and the other immune cells, produce IFN-, which could contribute to IRI directly by action on tubule cells and/or by activation of macrophages (6). Although adoptive transfer of CD4⁺ T cells from wild-type (WT) into RAG-1^–/– mice reconstituted injury following IR, adoptive transfer of CD4⁺ T cells from IFN- knockout (KO) mice did not (5). These results suggest that IFN- released by CD4⁺ T cells plays a critical role in the early pathogenesis of IRI.

Recently, NKT cells have been shown to participate in hepatic IRI (7, 8, 9). Two types of NKT cells (type I and II) represent a subset of CD4⁺ T cells, which possess features enabling their participation in the innate response since they can modulate Th cell differentiation and dendritic cell function (10). The phenotype of NKT cells is distinct from conventional T cells. The majority of NKT cells, referred to as type I NKT cells, express a V14-J18 rearrangement in mice, V24-JQ in humans and coexpress V8.2, V2, or V7. Unlike conventional T cells, NKT cells are activated by exogenous and endogenous glycolipid presented by the class I-like molecule CD1d (11, 12) and produce both Th1-type cytokines, IFN- and IL-2 and Th2-type cytokines, IL-4 and IL-10, within 1–2 h after activation (10). Type I NKT cells are mostly CD4⁺ but some are CD4^–CD8^–. Another class of NKT cells, referred to as type II NKT cells, is also CD1d-restricted and these cells do not express V14i TCR. These cells were identified based on the fact that NKT cell function was still detectable in mice lacking type I NKT cells but not in mice lacking CD1d. These two types of NKT cell populations may have distinct functions. The rapid response by NKT cells to express cytokines is in contrast to activation of conventional naive CD4⁺ cells which take 2–3 days (13, 14). These properties of NKT cells make them ideal candidates to participate in the early innate immune response to IR and could serve as a link to the adaptive immune response. Although the role of NKT cells in renal IRI is still unknown, an increase in CD3^intIL-2R cells, which are very closely related to the NKT cells was observed in the clamped kidney, liver, and spleen at 3 h (15).

Many studies have demonstrated that neutrophil infiltration of kidney tissue occurs as early as 4 h following IRI and reaches a maximum at 24 h (3). Neutrophils contribute to cytotoxicity through conventional activities including phagocytosis, chemotaxis, and oxidative burst. Accumulating information indicates that neutrophils play a more dynamic role responding to immune modulation. The recent finding demonstrating that V14i NKT can trigger IFN- production from neutrophils in early islet graft rejection (16) suggested the potential novel regulatory function of neutrophils by NKT cells in kidney IRI.

The current studies were aimed at determining the contribution of CD4⁺ cells as well as NKT cells to early IRI. Our results demonstrated that CD4⁺ cells infiltrate the kidney within 30 min following IRI. CD1d-restricted NKT cells were identified early in IRI and these cells produce a significant amount of IFN-. In addition, marked infiltration of GR-1⁺CD11b⁺ neutrophils into the kidney was observed following IRI and these cells produced much more IFN- compared with NKT cells as early as 3 h after reperfusion. A CD1d mAb which blocks the interaction between APCs and NKT cells or a mAb which depletes NKT cells attenuated renal IRI. Mice deficient in NKT cells (J18^–/–) were protected from kidney IRI. These three separate maneuvers that interfered with the APC-NKT axis attenuated NKT cell activation, neutrophil infiltration, and IFN- production by GR-1⁺ neutrophils. These results suggest that IR-induced activation of CD1d-restricted NKT cells leads to the subsequent recruitment and production of IFN- by neutrophils and kidney tissue injury.

	Materials and Methods

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Surgical protocol

All animals were handled and procedures were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We used C57BL/6 male mice (20 g between 6 and 8 wk of age; Charles River Laboratories). J18^–/– mice were provided by Dr. M. Taniguchi (RIKEN Research Center for Allergy and Immunology, Department of Molecular Immunology, Chiba University, Chiba, Japan). Bilateral flank incisions were performed as previously described (5). Mice were anesthetized with a regimen that consisted of ketamine (100 mg/kg, i.p.), xylazine (10 mg/kg, i.p.), and acepromazine (1 mg/kg, i.m.) and were placed on a thermoregulated pad to maintain body temperature at 37°C. Both renal pedicles were exposed and cross-clamped for 32 min then kidneys were reperfused (by releasing the clamp) for different time periods (0.5, 1, 3, 6, and 24 h). Control, sham-operated mice underwent a similar procedure as mice in the experimental group, however the renal pedicles were not clamped.

Blocking NKT cell activation or depleting NKT cells

A CD1d mAb (clone 1B1) (17) was used to block NKT cell activation. In preliminary studies, we administered the CD1d-blocking mAb (50, 150, or 300 µg) or rat IgG2b isotype control i.p. 24 h before kidney IR and at the onset of reperfusion. The increase in plasma creatinine following kidney IR in mice treated with IgG2b was reduced in mice treated with the CD1d mAb in a dose-dependent manner by 60, 70, and 80%, respectively. Subsequent studies used 50 µg of CD1d mAb or 50 µg of IgG2b isotype control. Blood was obtained and kidneys were harvested after 3 and/or 24 h of reperfusion.

An NK1.1 mAb (clone PK136) was used to deplete NKT cells. In preliminary studies, we administered, in normal mice that did not undergo IR, NK1.1 mAb (50, 100, or 200 µg) or control IgG2a (eBioscience) i.v. and harvested kidneys, liver, and spleen 48 h later. We observed that 100 and 200 µg of the NK1.1 mAb depleted NKT cells completely from all three organs; 50 µg of NK1.1 mAb had a similar effect but a small residual amount of NKT cells (<5%) remained in the spleen. In subsequent kidney IRI experiments, we administered i.v. 50 µg of NK1.1 mAb or 50 µg of IgG2a isotype control 48 h before bilateral renal pedicle clamping.

Kidney tissue processing and cell counting by FACS

Kidney suspensions were prepared from mice subjected to IRI or sham operation. Kidneys were weighed, minced, and incubated with collagenase type IA (10 µg/ml; Sigma-Aldrich) in cold Dulbecco’s PBS buffer with EDTA (2 mM) for 15 min at 37°C. The digested kidney tissue suspension was teased through a 100-µm BD Falcon cell strainer (Fisher Scientific) via a rubber end of a 1-ml syringe plunger and then passed through a cotton column treated with 10% FCS and centrifuged at 1200 rpm/min for 10 min. The cell pellet was washed with 1% BSA in PBS containing 0.1% sodium azide (Sigma-Aldrich) and resuspended. All Abs, unless otherwise stated, were purchased from eBioscience. After blocking nonspecific Fc binding with anti-mouse CD16/32 (2.4G2), fresh kidney suspensions were incubated with anti-mouse CD45-FITC (30-F11) for 30 min on ice. Sample volume was adjusted based on the kidney weight (grams per milliliter). The kidney suspension (100 µl) was mixed thoroughly with 30 µl of Caltag Counting Beads (994 beads/µl) before acquisition by BD FACSCalibur (BD Biosciences). Caltag Counting Beads (Caltag Laboratories-Invitrogen Life Technologies) were used to normalize for differences in cell recovery among samples. At least 1000 bead events over 2 min were acquired to insure the accuracy of the assay. CD45 cell absolute count (g^–1 kidney) = (events of CD45 cells counted/total number of beads counted (A+B) x input bead number)/g kidney. The leukocyte subset cell number (g^–1 kidney) was multiplied by the CD45 cell number and by the percentage of the subset. For example, CD4 T cell number (g^–1 kidney) = total CD45 cell number x percent of CD4 gated on the CD45 population. The remaining kidney suspension was labeled with anti-mouse CD4-allophycocyanin (L3T4), anti-mouse CD69-PE (H1.2F3), and anti-mouse LFA-1-PE (M17/4).

Identification of NKT cells and quantitation of intracellular IFN-

Cells were blocked with 2.4G2 (anti-mouse FcR II/III, 10 µg/ml) before all subsequent labeling and 7-aminoactinomycin D (7-AAD; 2 µg/ml; Invitrogen Life Technologies-Molecular Probes) was added to the surface labeled samples 15 min before running the samples to distinguish between live (7-AAD negative) and dead cells (7-AAD positive). Subsequent flow cytometry data acquisition was performed on BD FACSCalibur and analyzed by Flowjo software 6.4 (Tree Star). Ab concentration used in flow cytometry was 5 µg/ml unless otherwise noted. To identify CD1d-restricted NKT cells, we labeled kidney cells with anti-mouse CD45-FITC, anti-mouse TCR-PE, and CD1d tetramer-Alexa 647 loaded with PBS57 (1:500), an analog of -galactosyl ceramide (GalCer) that yields results identical with GalCer (National Institutes of Health tetramer facility; www. yerkes.emory.edu/TETRAMER/CD1d_Tetramers.html) (18). Unloaded CD1d tetramer-Alexa 647 was used as control. Intracellular IFN- labeling of NKT cells and neutrophils was done in parallel with cell surface labeling using anti-mouse CD45-FITC or PE, CD1d tetramer-Alexa 647, anti-mouse GR-1 (Ly6C/G or Ly6G)-PE, anti-mouse 7/4-PE, anti-mouse CD11b-PE, and anti-mouse Ly6C-FITC (AL-21; BD Biosciences). After fixation with 4% paraformaldehyde containing 7-AAD (10 µg/ml) for 30 min on ice and being washed with 1% BSA/PBS/0.1% azide (labeling buffer), cells were permeabilized for 30 min on ice with 0.3% saponin in labeling buffer containing 5% nonfat milk and 7-AAD. The washed cells were finally incubated with anti-mouse IFN--PE (XMG1.2, 10 µg/ml) in labeling buffer with 7-AAD and saponin. Cells were washed by using 0.3% saponin/10 µg/ml 7-AAD in labeling buffer, and IFN- production in NKT cells, as well as neutrophils, was measured by four-color FACS. Appropriate fluorochrome-conjugated, isotype-matched, irrelevant mAbs were used as negative controls.

Analysis by ImageStream 100 imaging flow cytometry of IFN- produced by GR-1⁺ cells infiltrating kidneys subjected to IRI

We used anti-CD45 microbeads (Miltenyi Biotec) to isolate CD45⁺ cells from kidneys subjected to IRI according to the manufacturer’s protocol. IFN--producing neutrophils were labeled with anti-mouse CD45-PE-Cy5 and anti-mouse GR-1-FITC followed by fixation and intracellular labeling with anti-IFN--PE. Cells were washed and suspended in 7-AAD. Images were collected using ImageStream 100 imaging flow cytometry (Amnis Corporation). Single-color control was also performed at the same time. A total of 10,000 events of GR-1⁺ cell images were collected. IDEAS Image Analysis Software (Amnis Corporation) was used for data analysis.

Histochemistry

Mouse kidneys were fixed in periodate-lysine-paraformaldehyde (4% paraformaldehyde) and embedded in paraffin, and 4-µm sections were cut. Sections were subjected to routine staining with H&E and viewed by light microscopy (Zeiss AxioSkop) under x200 magnification. Photographs were taken and brightness/contrast adjustment was made with a SPOT RT camera (software version 3.3; Diagnostic Instruments).

Statistics

GraphPad Instat 3 and GraphPad Prime 4 were used to analyze the data. Values were mean ± SEM. Unpaired and paired t test were performed as appropriate. A value of p < 0.05 was used to indicate significance.

	Results

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Kidney leukocyte population identification

We gated on CD45⁺ (FL1; Fig. 1a) and 7-AAD-negative cells (FL3; Fig. 1b) to identify live leukocytes. CD4⁺ cells (Fig. 1c) and CD1d-restricted NKT cells (TCR⁺CD1d tetramer⁺) (Fig. 1d) were found in this population. All CD1d tetramer-positive cells were TCR positive. TCR⁺CD1d⁺ cells represent 5–7% of the CD45 population in sham kidney.

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Kidney leukocyte subpopulations identified by four-color FACS. Kidney cell suspensions were prepared from sham-operated mouse kidneys and leukocyte subsets were identified as described in Materials and Methods. A CD45 mAb was used to identify the total leukocyte population (a) and 7-AAD was used to distinguish live cells (7-AAD negative) from dead cells (7-AAD positive) (b). Gating on the viable leukocyte population, we identified CD4+ (c) or CD1d tetramer-positive CD1d-restricted NKT cells (d). Numbers in each quadrant or delineated region represent the percentage of each cell type contained within the CD45+7AAD– population. SSC, side scatter; FSC, forward scatter.

The kidney content of CD4⁺ cells increased by 45% above sham within 30 min following IRI (p < 0.005, n = 15), peaked at 1 h (87% increase above sham; p < 0.01; n = 6) and persisted at 24 h of reperfusion (33% above sham; p < 0.001; n = 15; Fig. 2a). Because the kidney content of CD4⁺ cells increased within 30 min of reperfusion, we sought to characterize the activation state of CD4⁺ cells. Following IRI, there was a significant increase in the number of CD4⁺CD69⁺cells within 0.5 h of reperfusion (132% increase above sham; p < 0.05; n = 11) that persisted for 24 h (Fig. 2b). Leukocyte adhesion to the endothelial cells is important in the initiation of IRI. Therefore, we also examined the adhesion marker, LFA-1, expressed on the CD4⁺ cells. The number of CD4⁺LFA-1^high cells increased after 30 min of reperfusion (180% increase above sham, p < 0.05, n = 6), peaked at 1 h, and remained elevated for 24 h (Fig. 2c). These results indicate that there is an increase in kidney infiltration of total and activated CD4⁺ T cells as early as 30 min following IRI.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Early IRI leads to activation of CD4+ cells and enhanced expression of leukocyte adhesion molecule. Cell suspensions were prepared for flow cytometry from kidneys of mice subjected to sham () operation or renal IR () at 0.5, 1, and 24 h post ischemia as described in Materials and Methods. Counts are shown for total CD4+ cells (a), CD4+CD69+ cells (b), and CD4+LFA-1high cells (c). Values are means ± SE and represent total leukocyte counts (g–1 kidney). n = 4–15 for all groups. Symbols represent significant difference from sham: *, p < 0.05; , p < 0.01; , p < 0.005; and , p < 0.001.

IRI leads to early CD1d-restricted NKT cell activation and the production of IFN- by NKT cells

We found an early (30 min) increase in kidney content of activated CD4⁺ cells following reperfusion. CD1d-restricted NKT cells, a subset of CD4⁺ cells, also participate in innate immunity and early activation of NKT cells may contribute to early initiation of IRI. We quantitated IFN--producing CD1d-restricted NKT cells by using CD1d tetramer loaded with PBS57 (an analog of GalCer with high affinity and specificity for V14) and by measuring the expression of intracellular IFN-. We found that the number of CD1d-restricted NKT cells in kidney did not increase following 32 min of ischemia and 3 h of reperfusion (Fig. 3, a and b; Table I). However, there was an increase in the percentage of NKT⁺IFN-⁺ cells relative to gated NKT cells (Fig. 3c; Table I) of 27.28 ± 3.76 (n = 4) and 46.85 ± 0.91 (n = 5) for sham and IRI, respectively (p < 0.05).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. CD1d-restricted NKT cell expression of intracellular IFN- following IRI. Mouse kidneys were subjected to sham or 32 min ischemia followed by 3 h (a–c) or 24 h reperfusion (d–f) and intracellular IFN- was detected in CD1d-restricted NKT cells (CD1d tetramer-Alexa 647+ cells) by flow cytometry. Gating on CD1d tetramer+ NKT cells at 3 h (c) and 24 h (f) post ischemia, representative tracings of CD1d-restricted NKT cells expressing intracellular IFN- are shown for sham operation (blue line), IRI (green line), or isotype control (red line). % of Max, percentage of CD1d cells that are IFN-+; n = 4–7 for each group.

View this table: [in this window] [in a new window]   Table I. CD1d-restricted NKT cell and NKT+IFN-+ cells at 3 and 24 h following IRIa

IRI induces IFN- production by polymorphonuclear neutrophils (PMNs)

Interestingly, we observed that there were many CD1d tetramer^– cells expressing intracellular IFN- (Fig. 3, a and d, upper left quadrant) in sham-treated mice, which increased following 3 and 24 h of reperfusion (3- to 4-fold of sham; Fig. 3, b and e). We gated on the kidney CD45 population to determine the leukocyte subset expressing intracellular IFN-. The large percentage of CD1d tetramer^– IFN-⁺cells were not CD3⁺ T cells, DX5⁺ NK cells, macrophages (F4/80⁺), or dendritic cells (CD11c⁺ or IA⁺; Fig. 4, top panels). However, IFN- was expressed in the GR-1^high, 7/4⁺, CD11b^high, and Ly6C^int population, a phenotype consistent with neutrophils (Fig. 4, bottom panels). To exclude the possibility that we were detecting IFN- bound to IFN- receptors on the surface of leukocytes, we performed control experiments in which leukocyte labeling was performed in the absence of permeabilization. In the absence of permeabilization, IFN- labeling of PMNs was not found (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Neutrophils are the major IFN- producing cells. Mouse kidneys were subjected to 32 min of ischemia followed by 3 h of reperfusion. Anti-mouse CD3, DX5, F4/80, CD11c, MHC class II (IA) (a), Gr-1(Ly6C/G), 7/4, CD11b, and Ly6C (b) Abs were used to quantitate IFN- expression by leukocytes. Most of the IFN-+ cells were found in populations of GR-1high, ERRch 7/4, CD11bhigh, and Ly6Cint cells (b). Shown are representative examples from experiments repeated four times.

View larger version (48K): [in this window] [in a new window]   FIGURE 5. Morphology of IFN-+GR-1+ cells from reperfused mouse kidneys using ImageStream 100 flow cytometry. Mouse kidneys were subjected to 32 min ischemia followed by 3 h of reperfusion. Singlets were gated from the CD45+ kidney leukocyte population (a) and three representative images each of GR-1+IFN-+ (b) and GR-1+IFN-– cells (c) (from the 10,000 event file) were identified. The images of each cell, displayed in four of the available spectral channels, show BF illumination (gray) and fluorescent labeling with GR-1-FITC (green), IFN--PE (orange), and 7-AAD (nuclear labeling, red). The composite images represent an overlay of the images from the three fluorescent channels (FITC, PE, and 7-AAD).

View larger version (32K): [in this window] [in a new window]   FIGURE 6. IFN- expressed in GR-1+ cells from mouse kidney following 3 and 24 h of reperfusion. Mouse kidneys were subjected to sham or 32 min ischemia followed by 3 h (a–c) or 24 h of reperfusion (d–f). Intracellular IFN- expressed in GR-1+ cells were identified following 3 h of reperfusion in mice subjected to sham operation (a) or IRI (b). IFN- expression in GR-1+ cells at 24 h after reperfusion in mice subjected to sham (d) or IRI (e). We gated on GR-1+ cells from kidneys following 3 h (c) or 24 h (f) of reperfusion. Representative tracings are shown of neutrophils expressing intracellular IFN- from kidneys of mice subjected to sham operation (blue line), IRI (green line) isotype control (red line) are shown; n = 10–11 for each group.

View this table: [in this window] [in a new window]   Table II. Changes of GR-1+-producing IFN-+ cells at 3 and 24 h following IRIa

CD1d-dependent Ag presentation to NKT cells is required for NKT and neutrophil IFN- production

Our previous data showed the importance of IFN--producing CD4⁺ T and/or NKT cells in inducing neutrophil infiltration and kidney injury following IR (5). To determine whether CD1d-dependent Ag presentation is required for NKT cell production of IFN-, we used a CD1d mAb (1B1) to block the interaction between APCs and CD1d-restricted NKT cells. The CD1d mAb did not reduce the number of NKT cells expressing IFN- at 3 h following reperfusion (Fig. 7, a–c; Table III). However, at 24 h of kidney reperfusion, the CD1d mAb significantly blocked the increase in CD1d-restricted NKT cells (22% of IgG2b) as well as the fraction of those that expressed IFN-⁺ (23% of IgG2b; Fig. 7, d–f; Table III).

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Blocking NKT cell activation attenuates kidney infiltration of NKT cells and percentage of NKT cells expressing IFN- following IRI. Mice were administered a CD1d mAb (50 µg, i.p.) or isotype control IgG2b (50 µg, i.p.) 24 h before ischemia followed by an additional 50 µg at the onset of reperfusion as described in Materials and Methods. Mice were sacrificed at 3 h (a–c) (n = 8 for each group) and 24 h (d–f) (n = 4–9 for each group) after reperfusion and kidneys were obtained to examine the number of CD1d-restricted NKT cells producing IFN- following treatment with isotype control IgG2b or anti-CD1d mAb. CD1d-restricted NKT cells at 3 h (c) and at 24 h (f) following reperfusion were gated and representative tracings of CD1d-restricted NKT cells expressing intracellular IFN- from kidneys of mice subjected to sham operation (blue line), IRI (green line), or isotype control (red line) are shown.

View this table: [in this window] [in a new window]   Table III. The effect of a CD1d mAb on kidney content of IFN--producing NKT cells and neutrophils following 3 and 24 h of reperfusiona

View larger version (35K): [in this window] [in a new window]   FIGURE 8. Blocking NKT cell activation attenuates kidney infiltration of GR-1+ cells and percentage of GR-1+ cells expressing IFN- following IRI. Mice were administered a CD1d mAb (50 µg, i.p.) or isotype control IgG2b (50 µg, i.p.) 24 h before ischemia followed by an additional 50 µg at the onset of reperfusion as described in Materials and Methods. Mice were sacrificed at 3 h (a–c) (n = 8 for each group) and 24 h (d–f) (n = 4–9 for each group) after reperfusion and kidneys were obtained to examine the number of GR-1+ cells producing IFN- following treatment with isotype control IgG2b or anti-CD1d. GR-1+ cells at 3 and at 24 h (f) following reperfusion were gated and representative racings of GR-1+ cells expressing intracellular IFN- cells in IgG2b-treated group (blue line) and CD1d mAb-treated group (green line) and isotype control group (red line) are shown.

View larger version (77K): [in this window] [in a new window]   FIGURE 9. Blocking NKT cell activation protects kidney from IRI. Mice were administered a CD1d mAb (50 µg, i.p.) or isotype control IgG2b (50 µg, i.p.) 24 h before ischemia followed by an additional 50 µg at the onset of reperfusion (a–c). Plasma creatinine level is shown for sham-operated mice and mice whose kidneys were subjected to IR and treated with PBS, IgG2b, and CD1d mAb following 24 h after reperfusion (a). H&E staining of the outer medulla of IgG2b- (b) and CD1d mAb-treated mice (c). Values are mean ± SE; n = 5, 1, 10, and 10 for sham, PBS, IgG2b and CD1d mAb groups, respectively. *, p < 0.001. In a separate set of experiments, mice were administered an NK1.1 mAb (50 µg, i.v.) or isotype control IgG2a (50 µg, i.p.) 48 h before ischemia (d–f). Plasma creatinine level is shown for sham-operated mice and mice whose kidneys were subjected to IR and treated with IgG2a or NK1.1 mAb following 24 h of reperfusion (d). H&E staining of the outer medulla of IgG2a- (e) and NK1.1 mAb-treated mice (f). Values are mean ± SE; n = 6 each for IgG2a and NK1.1 mAb group; *, p < 0.001. H&E stain of the kidney outer medulla is shown for IgG2a-treated (e) and NK1.1 mAb-treated mice (f). Arrows indicate tubule necrosis. Magnification, x200.

Depletion of NKT blocks neutrophil influx, neutrophil IFN- production, and reduces injury following kidney IRI

In WT C57BL/6 mice, we depleted NKT cells by using an NK1.1 mAb and subjected kidneys to 32 min of ischemia followed by 24 h of reperfusion. The increase in plasma creatinine following IR was significantly decreased in mice treated with NK1.1 mAb (27% of IgG2a). Plasma creatinine was 0.18 ± 0.02 (n = 10) and 0.66 ± 0.09 (n = 10; p < 0.001; Fig. 9d) for NK1.1 mAb and IgG2a, respectively. H&E staining showed less kidney injury in the NKT cell depletion group compared with IgG2a group (Fig. 9, e and f). In kidneys subjected to IRI, the NK1.1 mAb reduced kidney neutrophil content (32% of control; p < 0.05) and percentage of GR-1⁺IFN- ⁺ cells (35% of control; p < 0.01; Table IV).

View this table: [in this window] [in a new window]   Table IV. The effect of an NK1.1 mAb on kidney content of IFN--producing neutrophils following 24 h of reperfusiona

NKT cell-deficient J18^–/– mouse kidneys are protected from IRI

J18^–/– mice lack the -chain of the invariant TCR of NKT cells, and therefore lack invariant NKT cells. Similar to experiments using the anti-CD1d mAb and anti-NK1.1 mAb, J18^–/– mouse kidneys were protected from IRI. The increase in plasma creatinine observed in WT mice subjected to kidney IR was significantly lower in the J18^–/– mouse. Plasma creatinine was 0.70 ± 0.02 (n = 4) and 0.23 ± 0.02 mg/dl (n = 5; p < 0.001) for WT and J18^–/– mice, respectively (Fig. 10a). Reduced injury in the J18^–/– mice was associated with fewer GR-1⁺ neutrophils (18% of WT; n = 4; p < 0.05) and IFN--producing neutrophils (15% of WT; n = 4–5; p < 0.05) (Fig. 10, b and c). H&E-stained kidney sections showed less kidney tubular injury in J18^–/– mice compared with WT mice following IRI (Fig. 10d). These results strongly support the role of NKT cells and GR-1⁺IFN- in mediating kidney IRI.

View larger version (60K): [in this window] [in a new window]   FIGURE 10. Kidneys from J18–/– mice are protected from IRI. Kidneys of WT and NKT cell-deficient J18–/– mice were subjected to 32 min of ischemia and 24 h of reperfusion. Plasma creatinine level is shown for sham-operated mice, WT and J18–/– mice (a). Values are mean ± SE; n = 4–5 for each group. *, p < 0.001 compared with corresponding sham group, , <0.05 compared with WT IRI group. Using FACS, we gated on CD45+7AAD– live leukocyte population and found kidney GR-1+ neutrophils and IFN--producing neutrophils (GR-1+IFN-+) in WT and J18–/– mice subjected to sham and kidney IRI (b). The cell number (x10,000) of the GR-1+IFN-+ is shown (c). Values are mean ± SE; n = 4–5 for each group; , p < 0.01 compared with sham; , p < 0.01 compared with WT IRI group. H&E stain of the kidney outer medulla is shown for WT sham (d, top, left) and IRI (d, top, right) mice, J18–/– sham (d, bottom, left) and IRI (d, bottom, right) mice. Arrows indicate tubule necrosis. Original magnification, x200.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The role of immune cells in the inflammatory response following renal IRI is complex (3) but there is increasing evidence that they participate in early innate and late adaptive immunity following renal IRI (19, 20). A large body of evidence now points toward the important role that CD4⁺ cells play in early IRI of kidney (4, 5) and of other tissues (21, 22). The conventional paradigm in which CD4⁺ cells are activated by Ag presentation, a process that requires 2–3 days, is, however, inconsistent with the time course for injury following IRI. In our previous studies, we showed that CD4⁺ cells contribute to IRI within 24 h of reperfusion (5). An important prerequisite for CD4⁺ cells to contribute to early IRI is early kidney infiltration. In the current study, we observed by flow cytometry that within 30 min of reperfusion, the total number of CD4⁺ T cells and activated CD4⁺ T cells increase in injured kidneys. These results suggest that very early in the reperfusion phase CD4⁺ T cells transition from the resting state to an active state and that a greater number of CD4⁺ cells expressing adhesion molecules such as LFA-1 could facilitate leukocyte adhesion. This conclusion is consistent with the concept that CD4⁺ cells participate in early IRI through an Ag-independent process in which T cells are activated by free radicals and cytokines (19, 23).

In the current study, we identified CD1d-restricted NKT cells by CD1d tetramers loaded with -GalCer analog PBS57 (18). At 3 h following reperfusion, we observed an increase in the percentage of NKT cells producing IFN-. At 24 h after reperfusion, the total number of NKT cells and the percentage of IFN--producing NKT cells are increased. Because these cells are activated by the - GalCer analog and produce IFN-, we believe these cells represent type 1 NKT cells. Because APCs activate NKT cells by presenting glycolipids through CD1d, we determined the effect of anti-CD1d mAb, which prevents the interaction between NKT cells and APCs, on kidney IRI. When administered before IRI, the CD1d mAb reduced renal injury and the percentage and number of NKT cells producing IFN-. Using two additional strategies we observed similar results. Depletion of NKT cells with an NK1.1 mAb reduced IRI. Mouse NKT cells are primarily type I NKT cells (also referred to as iNKT cells) characterized by express an invariant TCR -chain rearrangement (V14-J18) with a conserved CDR3 region, and coexpress V8.2, V2, or V7. V24-J18 coexpress V11-containing the TCR -chain in humans (24). CD1d-restricted NKT cells are composed of invariant NKT cells and also a minor NK1.1-negative NKT cell population. J18-deficient mice lack of type I NKT cells. In addition to the mAbs used to deplete NKT cells or block APC-NKT interaction, kidneys of NKT cell-deficient J18^–/– mice were also protected. These complementary approaches that interfere with NKT cell activation reduced kidney injury, an effect likely mediated through attenuating neutrophil and IFN--producing neutrophil recruitment to the kidney. These data strongly endorse the role of the type I NKT cells as an important early trigger for inflammatory immune response of kidney IR.

Also, these results are consistent with and extend our previous studies demonstrating the importance of IFN- bone marrow-derived cells in mediating renal IRI (5). In these studies, we found that chimeric mice, generated by reconstituting WT mice with bone marrow from IFN- KO mice (IFN-^–/–WT chimera), showed reduced renal IRI when compared with control chimeras (WTWT chimera). In addition, the renal protection observed in RAG-1^–/– mice subjected to kidney IRI was reconstituted with adoptive transfer of CD4⁺ cells from WT but not from IFN- KO mice. These results demonstrate the causal relation between CD4⁺ IFN- production and IRI. CD4⁺ cells are not only comprised of Th cells but also NKT cells. Thus, the increase in both total number of NKT cells and production of IFN- by NKT cells and the attenuation through CD1d Ag presentation blockade using an anti-CD1d Ab point to an important pathogenic role of type 1 NKT cell IFN- in IRI. This conclusion is also supported by similar kidney tissue protection observed in mice deficient of NKT cells. We observed similar tissue protection from kidney IRI in mice in which a mAb was used to deplete NKT cells or in mice deficient of NKT cells (J18^–/–). In all cases, when the APC-NKT axis was interrupted there was an attenuation of NKT cell activation, neutrophil infiltration, and IFN- production by GR-1⁺ neutrophils.

The CD1d mAb can block NKT cell activation by binding to the CD1d molecule of APCs. In our experiments, reduced IFN- production was detected because NKT cells cannot become activated without the CD1d-glycolipid-TCR complex interaction. At an early time point of renal IRI, we demonstrated an increase in the influx of CD4⁺ T cells and NKT cells. Chemokines mediate leukocyte migration into the injured kidney and IFN- modulates chemokine production, including IFN--inducing protein 10 (IP-10), monokine-induced by IFN-, and IFN--inducible T cell chemoattractant production, which bind to CXCR3 to mediate T cell migration (25) (25, 26, 27, 28, 29, 30). Our previous study showed that renal IRI up-regulated IP-10 and MIP, MIP1, MIP2, which are thought to mediate T cell and neutrophil migration (31, 32). The source of increased IFN- production upon stimulation by IL-12 is mainly from Th1, NKT (14, 33, 34), and myeloid cells (35). In renal IRI, in situ activation of Th1 and NKT cells may participate in mediating T cell influx by inducing IP-10 and IFN- production (31, 36). We observed that blocking NKT cell activation reduced IFN- production, which then led to a decrease in subsequent NKT cell and neutrophil recruitment.

The signaling pathway that links NKT activation to IFN- production from neutrophils is not known. We do know, however, that IFN- produced by activated NKT cells can activate CD8⁺ and NK cells to produce IFN- and dendritic cells to produce IL-12 (14). NKT cell activation in vivo by GalCer increased production of neutrophil IFN- (16). Other studies found IFN- can activate neutrophils to facilitate their release through degranulation of O₂ and preformed IFN- (35). Furthermore, IL-12 induces IFN- production by PMNs (35). The observation that PMNs secrete preformed IFN- and synthesize de novo IFN- to act in a paracrine or autocrine fashion suggest that they may play a role in linking innate and adaptive immune responses (35). Other studies found that IFN- can activate release of free radicals and facilitate bacterial killing (37). PMNs are known to contain small stores of IFN- that are readily released by degranulation. Furthermore, IL-12 induces IFN- production by PMNs (35). IFN- in this role is thought to link the innate and adaptive immune responses.

We believe that our study demonstrates for the first time a potential role for IFN--producing neutrophils in acute renal IRI. We found that the majority of IFN-⁺ cells were not NKT cells but were neutrophils. Other studies have demonstrated that myeloid cells like macrophages and dendritic cells produce IFN- (38, 39, 40, 41, 42, 43, 44). However, few studies have shown that neutrophils produce IFN- (35, 45, 46, 47) and no study to date has demonstrated the potential role of neutrophil IFN- in acute IRI. IFN- is a type II IFN that is produced early by NK cells and later by CD4⁺ T cells to enhance the effectiveness of dendritic cells and macrophages in Ag presentation (48) following pathogen exposure. APCs regulate this process through the production of IL-12. Additionally, IFN- is also known to play a critical role in the activation of neutrophils even though this concept has been overlooked (49). Activated neutrophils can then secrete preformed IFN- and/or produce more IFN- through enhanced transcription (35). Thus, myeloid cells including neutrophils are an important source and target for IFN-. IFN- is an important immunoregulator and can activate macrophages and up-regulate gene expression via a Jak-Stat tyrosine kinase-dependent or -independent pathway (50) and increase oxidative burst. IFN- also affects leukocyte-endothelial cells interaction (6). Thus, neutrophil production of IFN- is consistent with the induction of important deleterious effects in kidney IRI. Because both NKT cells and neutrophils are components of innate immunity and because NKT cells interact with other immune cells such as lymphocytes, NK cells, dendritic cells, and macrophages, we believe that NKT cells interact and regulate neutrophils.

In summary: 1) as early as 30 min following IRI, the kidney content of activated CD4⁺ cells increases, 2) early in renal IRI, a subpopulation of CD4⁺ cells, NKT cells, is activated through interaction with APCs, 3) blocking the activation of NKT cells through interaction with anti-CD1d mAb reduces NKT and neutrophil IFN- production and rescues the kidneys from injury, and 4) NKT cell deficiency also inhibits neutrophil recruitment and its production of IFN-. We conclude that early innate immunity following IRI is due in part to Ag-dependent activation of NKT cells and subsequent neutrophil infiltration and IFN- production.

	Acknowledgments

 
We are grateful to Joanne Lannigen in Flow Cytometry Core Facility for assistance with the ImageStream 100 imaging flow cytometry data acquisition and analysis, Amy Vergis for technical assistance, and Drs. Joel Linden (University of Virginia, Charlottesville, VA), Alaa S. Awad (University of Virginia) and Diane Rosin (University of Virginia) and Luc Van Kaer (Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN) for helpful discussions. The anti-mouse CD1d mAb clone 1B1 was a gift from Dr. Mitchell Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA). The mAb PK136 was a gift from Dr. Wayne M. Yokoyama (Howard Hughes Medical Institute, Washington University, St. Louis, MO). J18^–/– mice were a gift from Dr. Masaru Taniguchi (RIKEN Research Center for Allergy and Immunology, Department of Molecular Immunology, Chiba University, Chiba, Japan). Tetramers were provided by the National Institutes of Health Tetramer Facility (Emory University, Atlanta, GA).

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by funds from National Institutes of Health Grants R01 DK56223, R01 DK62324, R01 H2070065, R01 CA78400, and R21 AI059996.

² Address correspondence and reprint requests to Dr. Mark D. Okusa, Division of Nephrology, Box 800133, University of Virginia Health System, Charlottesville, VA 22908. E-mail address: mdo7y{at}virginia.edu

³ Abbreviations used in this paper: IR, ischemia-reperfusion; IRI, IR injury; KO, knockout; 7-AAD, 7-aminoactinomycin D; -GalCer, -galactosyl ceramide; PMN, polymorphonuclear neutrophil; BF, bright field; WT, wild type; IP-10, IFN--inducing protein 10.

Received for publication August 3, 2006. Accepted for publication February 12, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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