transfused macrophages ameliorate pancreatic and renal injury in murine diabetes mellitus

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Original Paper

Nephron Exp Nephrol 2011;118:e87–e99 DOI: 10.1159/000321034

Transfused Macrophages Ameliorate Pancreatic and Renal Injury in Murine Diabetes Mellitus

Dong Zheng a Yiping Wang a Qi Cao a Vincent W.S. Lee a Guoping Zheng a Yan Sun a Thian K. Tan a Ya Wang a Stephen I. Alexander b David C.H. Harris a

a Centre for Transplant and Renal Research, University of Sydney, Westmead Millennium Institute, and b Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, N.S.W. , Australia

assessed by insulin staining, haemoglobin A1c and blood glucose was reduced after transfusion of M2 macrophages. In vivo, activation of kidney endogenous macrophage cyto-kine expression was inhibited by M2 macrophages. Conclu-

sion: Our findings show that M2 macrophages can protect against islet and renal injury in streptozotocin-induced dia-betes, providing a potential therapeutic strategy for diabetes and diabetic nephropathy. Copyright © 2011 S. Karger AG, Basel

Introduction

In human and animal diabetic nephropathy, the ac-cumulation of macrophages in the kidney is linked to the degree of histological and functional injury, suggesting a pathogenic role for macrophages [1–3] . Activated effector macrophages can increase renal inflammation and exac-erbate renal injury by homing to the damaged kidney [4] . Macrophage recruitment into diabetic kidneys is direct-ed by several chemokines including CCL2, CX3CL1, CCL5 and osteopontin [5, 6] . CCL2, a chemokine possi-bly induced by hyperglycemia in diabetes, plays a key role

Key Words

Macrophages � Cell transfer � Diabetic nephropathy

Abstract

Background: Alternatively activated macrophages (M2 mac-rophages) are able to reduce renal injury in murineadriamycin nephropathy. However, the effect of M2 macro-phages in other renal diseases such as diabetic nephropathy remains unknown. Methods: Macrophages were separated from splenocytes and polarized with IL-4 and IL-13 into a pro-tective phenotype. Mice underwent adoptive transfer with M2 macrophages, and then diabetes was induced by tail vein injection with streptozotocin (STZ). Blood glucose levels were monitored daily. Mice were sacrificed at week 10 after STZ. Renal function and histopathological injury were as-sessed quantitatively. Results: Transfused M2 macrophages accumulated progressively in kidneys for up to 10 weeks af-ter STZ. Kidneys from diabetic mice transfused with M2 mac-rophages had less tubular atrophy, glomerular hypertrophy and interstitial expansion than did control diabetic mice. M2 macrophages suppressed the development of interstitial fi-brosis. In addition, the degree of pancreatic islet injury, as

Published online: February 11, 2011

Dong Zheng Centre for Transplantation and Renal Research The University of Sydney at Westmead Millennium Institute Westmead, NSW 2145 (Australia) Tel. +61 2 9845 6346, E-Mail dzhe8849 @ mail.usyd.edu.au

© 2011 S. Karger AG, Basel1660–2129/11/1184–0087$38.00/0

Accessible online at:www.karger.com/nee

http://dx.doi.org/10.1159%2F000321034

Zheng /Wang /Cao /Lee /Zheng /Sun /Tan /Wang /Alexander /Harris

Nephron Exp Nephrol 2011;118:e87–e99 e88

in macrophage infiltration in diabetic nephropathy. The mechanisms by which macrophages cause renal injury are not fully understood, but are related to their secretion of various pro-inflammatory and pro-fibrotic mediators.

Macrophages are a diverse population of cells which can be polarized into effector macrophages (classically ac-tivated macrophages, M1) and suppressive macrophages (alternatively activated macrophages, M2) in vitro [7] . Thus, macrophages are no longer regarded merely as ef-fectors of injury, but also as potential therapeutic tools that can regulate inflammation. A major challenge is to determine how to best manipulate macrophage activation for their therapeutic use. Several approaches have been described, including ex vivo modulation of macrophage activation with cytokines and genetic manipulation. Mac-rophages transduced by specific transgenes to express IL-4 or IL-10 attenuated inflammation and reduced renal injury in rat nephrotoxic nephritis [8, 9] . Wilson et al. [10] reported that macrophages in which the NF- � B pathway was inhibited by transduction with dominant-negative I � B developed anti-inflammatory properties and reduced glomerular injury in nephrotoxic serum nephritis. De-spite genetic manipulation of macrophages being applied successfully in experimental models of renal disease, ma-jor obstacles to clinical application of this technology in-clude the danger of viral vectors and difficulty of control-ling levels of gene expression in vivo [11] . Macrophages modified ex vivo with IFN- � into M1 and infused into leucopenic rats substantially augmented renal injury, an effect which could be blocked with glucocorticoids [12] . However, until now there have been few reports regarding the use of alternatively activated macrophages (M2 mac-rophages) in vivo to prevent injury in renal disease.

In mice with adriamycin nephropathy, we have shown that M2 macrophages could protect against both struc-tural and functional damage [13] . Whether M2 macro-phages can protect against injury in diabetic mice is un-known. Here we investigated the possibility that macro-phages, modulated to a protective phenotype by IL-4 and IL-13, could protect against diabetes and renal injury in streptozotocin (STZ)-induced diabetic mice.

Methods

Streptozotocin Murine Model Six- to 8-week old male BALB/c mice, weighing approximate-

ly 22 g, were purchased from the Animal Resources Centre (Perth, W.A., Australia). The Animal Ethics Committee of Westmead Hospital approved the experimental procedures. Dose-finding studies defined an optimal regimen of two doses of STZ (75 and

150 mg/kg; Sigma Aldrich, Steinheim, Germany). The doses of STZ were injected 1 day apart via the tail vein of each nonanes-thetized BALB/c mouse. Mice were monitored daily for blood glu-cose and body weight. Haemoglobin A 1c (HbA1c), a marker of protein glycation, was determined from cardiac blood at 10 weeks.

Macrophage Separation from Spleen and ex vivo Culture of M2 Macrophages Splenocytes were harvested from BALB/c mice and washed in

cold RPMI 1640 medium (Invitrogen, Mount Waverly, Vic., Aus-tralia). Tissue was triturated with sterile syringes, and the result-ing cell suspension was filtered through a 40- � m nylon mesh (BD Biosciences, N.J., USA) and then incubated at 37 ° C for 30 min. Adherent cells were harvested and purified by MACS CD11b-pos-itive MicroBeads kit (Miltenyi Biotec, Bergisch Gladbach, Ger-many). These spleen-derived macrophages were rinsed three times with RPMI 1640 medium. To produce M2 macrophages, macrophages were incubated with IL-4/IL-13 (IL-4/IL-13, 10 ng/ � l each, Invitrogen, Carlsbad, Calif., USA) for 48 h. Unstimu-lated macrophages (M0) were used as controls.

Examination of Transfused and Endogenous Cells To isolate endogenous macrophages from kidney, cells were

stained with FITC-conjugated anti-mouse CD11b. Transfused macrophages (M2 macrophages, CD11c+DiI+) and endogenous macrophages (CD11b+DiI–) were sorted by FACS. Sorted cells were used for real-time PCR or FACS analyses to detect pheno-typic changes.

Quantitative Reverse-Transcription PCR RNA was isolated and reverse-transcribed with cDNA Synthe-

sis Kit (Invitrogen), and real-time PCR using the SYBR Master-mix (Invitrogen). The methods of analysis have been described previously [14] . Real-time PCR was carried out in two steps: step 1, 94 ° C for 10 min, 1 cycle, and step 2, 94 ° C for 15 s, followed by 60 ° C for 60 s, 45 cycles. Beta-actin was used as an endogenous reference to correct for differences in the amount of total RNA added to the reaction and to compensate for different levels of in-hibition during the reverse transcription of RNA and during PCR. Primers used in this study included: TNF- � 5 � to 3 � forward: GCTGAGCTCAAACCCTGGTA, reverse: CGGACTCCGCAA-AGTCTAAG; iNOS 5 � to 3 � forward: CACCTTGGAGTT CACC-CAGT, reverse: ACCACTCGTACTTGGGATGC; IL-12 5 � to 3 � forward: AGGTCACACTGGACCAAAGG, reverse: TGGTTTG-ATGATGTCCTGA; beta-actin 5 � to 3 � forward: GATTACTGCT-GGCTCCTAGCA, reverse: GCCACCGATCCACACAGAGT.

Macrophage Labeling, Adoptive Transfer and Tracking For in vivo tracking, M2 macrophages were stained with a red

fluorescent membrane label, DiI (Molecular Probes, Oreg., USA) according to the manufacturer’s instructions, and harvested into serum-free medium immediately before injection. DiI is a lipo-philic membrane stain that diffuses laterally to stain the entire cell. In brief, 50 � g DiI was dissolved into 50 � l pure ethanol. To a one-million-cell suspension in 1 ml RPMI 1640, 4 � l DiI was added for 10 min incubation at 37 ° C followed by 15 min incuba-tion at 4 ° C. 1 ! 10 6 cells were transfused by a single tail vein in-jection at day 0 followed by the first STZ injection. Fifty mice were caged according to groups: 26 (7 diabetic-M2, 7 diabetic-M0, 7 diabetic control, 5 normal) mice were sacrificed in week 10 after

Macrophages Ameliorate Diabetic Nephropathy


STZ injection for tissue examination, and 24 mice were sacrificed (diabetic-M2) on day 1, day 2, week 2 and week 10 after STZ injec-tion for M2 macrophages tracking.

Histology Kidneys were rapidly removed on day 70. Coronal sections of

renal tissue were immersion-fixed in 10% neutral-buffered for-malin and embedded in paraffin. 5- � m-thick sections were stained with periodic acid-Schiff (PAS) or trichrome. To quanti-tate tubular atrophy, the tubule cell height of an individual corti-cal tubule was measured using line morphometry (magnification ! 200) by computer image analysis software (Image J) [15] . The tubular diameter was defined as the length of a straight line that passed through the centre of a symmetrically sectioned tubule and joining two points on the tubular circumference. A total of 50 randomly selected cortical tubules in 10 nonoverlapping fields (magnification ! 200) were measured, and the mean cross-sec-tional tubule cell height was determined for each section. The cortical interstitial volume included the tubular basement mem-brane and peritubular capillaries. To quantitate this area, cortical fields (magnification ! 200) were viewed on a video screen; the area of interstitial space was determined with image analysis soft-ware and was expressed as a percentage of the total area of the field. The mean percentage area of five non-overlapping cortical fields was calculated for each section. Interstitial fibrosis wasassessed and quantified on trichrome-stained sections by point counting using Image J software in each of 10 non-overlapping randomly-selected cortical fields. Points falling within blue areas (fibrosis) were considered as positive. Scores derived from 10 fields per kidney section (3 sections/per mouse) were collected for the analyses. All histological data analyses were carried out in a blinded fashion.

Immunohistochemistry For immunohistochemical staining of macrophages, CD4+

and CD8+ cells, rat anti-mouse F4/80, CD4 and CD8 antibodies (BD Pharmingen, N.J., USA) were used as the primary antibody. Biotinylated peroxidase (Zymed, Calif., USA) and Streptavidin (Zymed) were used for the secondary antibody. Kidney sections were placed in OCT (Sakura Fintek Inc., Torrance, Calif., USA). Sections of 5 � m were cut, dried overnight and fixed in cold ac-etone for 8 min. Endogenous peroxidase activity was blocked by 0.3% (v/v) H 2 O 2 solution for 15 min when incubating the slides. Biotin Blocking System (Dako, Calif., USA) was used to block en-dogenous avidin-binding activity. Normal rat immunoglobulin was used for control sections. Sections were incubated with poly-clonal rabbit anti-rat Immunoglobulins/Biotinylated (Dako Cor-poration, Glostrup, Denmark) or RTU Vectastain Elite ABC Per-oxidase Kit (Vector Laboratories, Burlingame, Calif., USA), and 3,3-diaminobenzidine substrate chromogen solution (Dako) were applied and then washed. Slides were counterstained with hematoxylin (Sigma-Aldrich, Steinheim, Germany). For assess-ment of interstitial infiltration, positively stained cells located in the interstitial area were counted from more than 20 random cor-tical fields (magnification ! 200) in each section, and the num-bers averaged for each section. Pancreatic tissue was fixed and paraffin-embedded. Multiple slices were cut for insulin staining and quantitative evaluation. Tissue cuts were made at intervals of 100 � m to avoid counting any islet twice. Anti-guinea pig insulin antibody (Sigma, Germany) was used, and 20 random areas with

islet staining were photographed and size and intensity of insulin staining calculated in pixels by Image J.

Renal Function All urine and blood specimens were analyzed by the Institute

of Clinical Pathology and Medical Research (Westmead Hospital) using a BM/Hitachi 747 analyzer (Tokyo, Japan). 1 ml of heart blood was taken from each mouse and urine was collected for16 h using a special metal cage in the Department of Westmead Animal Care, Westmead Hospital.

M2 Macrophages Co-Culture with M0 Macrophages with High Glucose Media in vitro Splenic macrophages were seeded onto 13-mm-diameter plas-

tic coverslips (Nalge Nunc International, Rochester, N.Y., USA) and exposed to cytokines IL-13 and IL-4 for 48 h to become M2 macrophages, or cultured without cytokines for 48 h to remain as M0 macrophages. Splenic macrophages were stimulated with 30 mmol/l D -glucose (DG-30) (MP Biomedicals, Solon, Ohio, USA) or 30 mmol/l L -glucose (LG-30) (MP Biomedicals), or cultured with typical RPMI 1640 medium containing 10 mmol/l D -glucose (DG-10) as controls for 1, 4, 8 and 12 h. iNOS, TNF- � , IL-12 cy-tokine mRNA expression (relative to expression of housekeeping gene murine beta-actin) of these macrophages were examined. Macrophages were also grown in 24-well plates to confluent monolayer, and then cultured with M2 macrophages or with M0. The coverslips seeded with M2 macrophages were placed in direct contact with the macrophages in medium with 30 mmol/l D -glu-cose, or controls for 4 h.

Statistical Analysis The statistical package SPSS for windows version 14 was used

to analyze the data. Renal functional data (serum creatinine, cre-atinine clearance and urine protein) were log transformed before analysis to stabilize the variance. Spearman’s coefficient of rank was performed for the correlation analysis of data in and between treatment and control groups. Other data analysis was performed directly by one-way analysis of variance for multiple comparisons of parametric data. Results are expressed as the group mean 8 SD. p ! 0.05 was considered statistically significant.

Results

Induction of Diabetes in STZ-Treated Mice Within 8 days of STZ injection, all mice developed

overt diabetes. The mean blood glucose level was lower in diabetic-M2 compared to diabetic control mice and dia-betic-M0 mice on days from day 28 until the end of the study (p ! 0.01) ( fig. 1 a). The level of HbA1c at 10 weeks was significantly lower in diabetic-M2 macrophages mice compared to diabetic control mice and diabetic-M0 mice (diabetic-M2 vs. diabetic control vs. diabetic-M0: 3.20 8 0.07% vs. 4.38 8 0.63% vs. 4.92 8 0.73%, p ! 0.01) ( fig. 1 b).



Renal Histology and Function Renal damage at week 10 in diabetic control mice was

characterized by glomerular hypertrophy, tubular inju-ry and interstitial fibrosis with moderate intestitial leu-cocyte infiltration ( fig. 2 ). Kidneys from diabetic-M2 macrophages mice had less tubular injury (tubular cell height 11.5 8 0.5 � m, p ! 0.01), glomerular hypertrophy (3,273 8 220 � m, p ! 0.01) and interstitial expansion (25.9 8 2.3%, p ! 0.01) than control diabetic mice (9.7 8 0.3 � m, 3,798 8 249 � m and 35.4 8 1.1%) and dia-betic-M0 mice (9.7 8 0.6 � m, 3,872 8 352 � m and 34.3 8 2.1%). The degree of interstitial fibrosis as assessedby trichrome staining was also reduced in diabetic-M2 mice (normal vs. diabetic control vs. diabetic-M0 vs. di-abetic-M2: 0.2 8 0.09 vs. 4.3 8 0.92 vs. 4.1 8 0.81 vs. 2.2 8 0.42, p ! 0.01) ( fig. 2 ). There was no difference in glomerular sclerosis between diabetic-M2 and diabetic control mice (data not shown). Urinary protein excre-tion was assessed at ten weeks after STZ, and was sig-nificantly increased following administration of STZ (diabetic control vs. normal, 75 8 25 vs. 45 8 9 mg/16 h, p ! 0.05), but showed no improvement after trans-fusion of M2 and M0 macrophages (data not shown). Similarly, serum creatinine and creatinine clearance were significantly worse in diabetic control mice than normal (34 8 18 � mol/l and 0.08 8 0.05 ml/min vs. 18 8 1 � mol/l and 0.12 8 0.03 ml/min, p ! 0.05), but there

was no improvement after M2 and M0 macrophage transfusion (data not shown).

Insulin Staining of Islets To evaluate the degree of pancreatic injury, islets were

assessed in multiple slices by insulin staining. The insu-lin-staining area was dramatically reduced after admin-istration of STZ, but significantly less reduced in diabet-ic mice transfused with M2 macrophages as compared with diabetic control and diabetic-M0 (diabetic-M2 vs. diabetic control vs. diabetic-M0 14,790 8 2,580 vs. 2,322 8 1,053 vs. 2,098 8 999 pixels, p ! 0.01) ( fig. 2 a, b).

Relationship between Severity of Diabetes and Renal Injury We examined the relationship between the degree of

kidney injury versus the degree of pancreatic injury, and the extent of inflammatory cell infiltration of kidney ver-sus pancreas. There was no correlation between any of the functional and structural indicators of kidney injury and those of islet injury. In addition, the extent of inflamma-tion in pancreas did not correlate with that in kidney ( ta-ble 1 ) and there was no correlation between the indicators of renal injury and those of islet injury in diabetic control mice alone and among diabetic control and diabetic-M2 combined (data not shown).

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Fig. 1. a Blood glucose levels. Mean blood glucose level was significantly lower in diabetic-M2 than in diabetic control mice and diabetic-M0 from day 28 until the end of the study ( * p ! 0.01 vs. diabetic control and diabet-ic-M0). b HbA1c levels. Mean HbA1c was significantly lower in diabetic-M2 macrophages than in diabetic con-trol and diabetic-M0 mice. * p ! 0.01 vs. diabetic control and diabetic-M0. Data represent the mean 8 SD, n = 7 per group.



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Fig. 2. a Effect of M2 macrophage transfusion on renal and pancreatic injury. Representative PAS-stained and trichrome (Tri)-stained sections of renal cortex at week 10 (original magnification ! 400), and representative insulin stained sections of pancreas at week 10 (original magnification ! 200). Glomerular hypertrophy, tubu-lar atrophy and islet injury were reduced in diabetic-M2. Arrows indicate glomerular hypertrophy and tubular atropy. (Figure 2 continuous on the next page.)

Table 1. S pearman-rank correlation coefficients between indicators of pancreatic injury and renal injury together with associated p value in parentheses

P ancreatic injuryHbA1c insulin staining macrophage infiltration CD4 infiltration CD8 infiltration

Serum creatinine –0.105 (0.86) –0.1 (0.87) 0 (1.0) –0.1 (0.87) –0.1 (0.87)Glomerulosclerosis 0.211 (0.73) 0.6 (0.28) –0.765 (0.71) –0.654 (0.15) –0.4 (0.51)Glomerular tuft area 0.158 (0.80) 0.2 (0.74) –0.3 (0.62) –0.7 (0.19) 0.3 (0.62)Interstitial volume 0.738 (0.16) –0.5 (0.39) –0.3 (0.62) –0.2 (0.75) –0.2 (0.75)Tubular cell height 0.158 (0.8) 0.2 (0.75) –0.3 (0.62) –0.7 (0.19) 0.3 (0.62)Interstitial fibrosis –0.527 (0.36) 0.8 (0.10) –0.3 (0.62) –0.5 (0.39) 0 (1)Macrophage infiltration 0.158 (0.8) 0.2 (0.75) –0.3 (0.62) –0.7 (0.19) 0.3 (0.62)CD4 infiltration –0.791 (0.11) 0.3 (0.62) 0.7 (0.19) 0 (1) 0.764 (0.23)CD8 infiltration –0.053 (0.93) –0.5 (0.39) 0.7 (0.19) 0.8 (0.11) 0.3 (0.62)

The re were no statistically significant associations between the degree of renal injury and the degree of pancreas injury or renal and pancreatic inflammatory cells. All p > 0.1.



Macrophage and T Cell Infiltration in Pancreas and Kidney Leucocyte infiltration in pancreas and kidney on day

1, day 2, week 2 and week 10 was examined using anti-F4/80, CD4 and CD8 antibodies. Macrophage and T cell accumulation within kidney glomerular and tubuloint-erstitial compartments reached a peak at week 2 and then decreased with disease progression in diabetic mice. The number of these cells was markedly reduced in diabetic

mice transfused with M2 macrophages compared to dia-betic control and diabetic-M0 mice at week 2 and week 10. Similarly, leucocyte infiltration around islets in pan-creas was greatest at week 2 and less at week 10. The num-ber of these cells was significantly reduced in diabetic mice transfused with M2 macrophages compared to dia-betic control and diabetic-M0 mice at weeks 2 and 10 ( fig. 3 ).

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Fig. 2. b Quantitation of renal and islet injury after M2 macrophage transfusion. Kidney injury was assessed quantitatively by Image J (see ‘Methods’). * p ! 0.01 vs. diabetic control and diabetic-M0. Data represent the mean 8 SD, n = 7 per group.



Tracking of Transfused Macrophages in vivo To examine the disposition of transfused macro-

phages, 1 ! 10 6 DiI-labelled M2 and M0 macrophages were injected via tail vein on the day before STZ admin-istration. Fluorescence microscopy showed that M2

( fig. 4 a) and M0 macrophages ( fig. 4 b) aggregated in the spleen immediately after transfusion, and then accumu-lated progressively both in kidney and in pancreas, reach-ing a peak at week 2. Transfused macrophages were still evident in pancreas, kidney and spleen at week 10.

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Fig. 3. a, b Quantitation of time course of leucocyte accumulation in renal tubulointerstitum and glomerular compartments. Repre-sentative images showing CD4+ cells, CD8+ cells, macrophages in the kidney at day 1, day 2, week 2, week 10 from diabetic con-trol, diabetic-M0 and diabetic-M2. c The number of macrophages, CD4+ cells and CD8+ cells in periphery of islets. Cell number was quantified by point counting. * p ! 0.01 versus counterpart from diabetic control group and diabetic-M0; ** p ! 0.05. Data repre-sent the mean 8 SD, n = 7 per group.

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M2 Macrophages Suppress High Glucose-Stimulated Macrophages There was an approximate 4-fold increase in iNOS and

IL-12 and 2-fold increase in TNF-a mRNA expression in macrophages cultured with glucose in high concentra-tion (30 mol/l D -glucose, DG-30) for 4 h ( fig. 5 a). Macro-phage iNOS and IL-12 and TNF-a mRNA expression was increased at one hour after DG-30 stimulation, peaked at 4 hours, and gradually reduced to the levels of non-stim-ulation up to 16 h ( fig. 5 b). When macrophages activated by DG-30 were co-cultured with M2 macrophages, mac-rophage mRNA expression of IL-12, TNF-a and iNOS was significantly reduced compared to control groups ( fig. 5 c).

Deactivation by M2 Macrophages of Renal Endogenous Macrophages in vivo In comparison to diabetic control and diabetic-M0

mice, the expression of IL-12, iNOS and TNF- � by en-dogenous renal macrophages (EM), which include both resident and infiltrating macrophages, in the diabetic-M2 group was considerably reduced at day 70 ( fig. 6 ).

Discussion

In this study, the effect of protective macrophages (M2 macrophages), which had been modulated ex vivo by cy-tokines IL-4 and IL-13, was examined in a model of dia-betic nephropathy. Renal structural injury in diabetic mice was significantly reduced by M2 macrophages, as evidenced by less glomerular hypertrophy, tubular atro-

phy and interstitial inflammation and fibrosis. Moreover, islet structural injury, HbA1c and glucose levels were re-duced significantly in diabetic mice transfused with M2 macrophages as compared to untransfused diabetic mice. There was a marked reduction of macrophages and CD4+ and CD8+ T cells in both renal cortex and pancreas of diabetic mice transfused with M2 macrophages.

Macrophages display great plasticity in response to various microenviromental stimuli. For example, it has been reported that macrophages in adipose tissue of non-obese humans are M2 macrophages in type [7] . However, the phenotype of macrophages in obese type II diabetics is pro-inflammatory [16] . Macrophages modulated ex vivo by IL-4 and IL-13 develop a protective phenotype and secrete suppressive cytokines such as IL-10 and TGF- � . Previously, we have transfused these alternatively acti-vated macrophages into mice as a treatment for adria-mycin-induced nephropathy (AN) [13] . AN is an inflam-matory renal disease analogous to human focal segmental glomerulosclerosis [17] . That study proved directly that M2 macrophages are able to protect against renal injury. Advantages of this method of producing protective mac-rophages include the simplicity of ex vivo macrophage modulation by cytokines, and its avoidance of the limita-tions of gene therapy including gene delivery and risks from the viral vector. In the current study, diabetic renal structural injury was also significantly improved by transfused M2 macrophages.

The anti-inflammatory effect of M2 macrophages could explain how M2 macrophages ameliorated renal injury. Inflammation has been demonstrated to be a crit-ical factor in the pathogenesis of diabetic nephropathy. Pro-inflammatory cytokines, including TNF- � and INF- � , are highly expressed in kidneys in STZ-induced diabe-tes [18] . In addition, chemokines such as CCL2 are se-creted by tubular cells in response to high glucose con-centrations, thereby attracting macrophages and T cells to the sites of injury. Macrophages and other leucocytes are increased substantially within glomeruli and renal in-terstitium in diabetic nephropathy and contribute to the irreversible structural damage [1] . Glomerular macro-phages have been shown to be capable of stimulating ma-trix expansion by producing and promoting mesangial cells to secrete TGF- � , and in turn inducing overproduc-tion of extracellular matrix proteins including type IV collagen [19, 20] . CD4+ and CD8+ T cells have also been reported to initiate IFN- � secretion in response to ad-vanced glycosylation end products, which would induce further renal inflammation and oxidative stress [21] . In the current study, the numbers of macrophages and T

Fig. 4. a DiI-stained transfused M2 macrophages in kidney, pan-creas and spleen (original magnification ! 200). Representative fluorescence photomicrographs showing M2 localization in pan-creas ( A–D ), kidney ( F–I ) and spleen ( K–N ) at day 1 ( A , F , K ), day 2 ( B , G , L ), week 2 ( C , H , M ), week 10 ( D , I , N ) and the corresponding negative control ( E , J , O) . b DiI-stained transfused M0 macro-phages in kidney, pancreas and spleen (original magnification ! 200). Representative fluorescence photomicrographs showing M0 localization in pancreas ( A–D ), kidney ( F–I ) and spleen ( K–N ) at day 1 ( A , F , K ), day 2 ( B , G , L ), week 2 ( C , H , M ), week 10 ( D , I , N ) and the corresponding negative control ( E , J , O) . c Quantitation of DiI-stained M2 macrophages accumulation in kidney, pancre-as and spleen. d Quantitation of DiI-stained M0 macrophages ac-cumulation in kidney, pancreas and spleen. Transfused M2 and M0 were quantified in spleen, pancreas and kidney. n = 6 at each time point, * p ! 0.01 vs. the other three time points and vs. day 1, day 2 and week 10, respectively. Data represent the mean 8 SD.



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Fig. 5. a High glucose effect on expression of IL-12, TNF- � and iNOS mRNA by macrophages. Macrophages were cultured with D -glucose 30 mmol/l (DG-30) for 4 h and total cellular mRNA was extracted for real-time RT-PCR analysis. Data represent the mean 8 SD of three experiments, run in triplicate. * p ! 0.01 vs. D -glu-cose 10 mmol/l (DG-10) and L -glucose 30 mmol/l (LG-30). Data represent the mean 8 SD of three experiments. b Expression of iNOS and inflammatory cytokines from macrophages exposed to high glucose. mRNA gene expression of iNOS, IL-12 and TNF- �

by macrophages exposed to D -glucose 30 mmol/l (DG-30) reached a peak at 4 h, subsequently decreased and returned to normal lev-el at 16 h. c The effect of M2 macrophages on expression of IL-12, TNF- � and iNOS mRNA by macrophages exposed to high glu-cose. M2 macrophages were co-cultured with glucose-activated macrophages (M0 DG-30) for 4 h. mRNA expression of cytokines was examined by real-time PCR. Data represent the mean 8 SD of three experiments, run in triplicate. * p ! 0.01 and * * p ! 0.05 vs. the other four time points.

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cells in diabetic kidneys were markedly increased at week 2 after STZ, as we have reported previously [22] . How-ever, in diabetic mice transfused with M2 macrophages, the numbers of macrophages and T cells were dramati-cally reduced at weeks 2 and 10. This reduction in leuco-cyte numbers was concordant with the demonstrated ac-cumulation of transfused M2 macrophages in kidneys within the first 2 weeks.

Macrophage-derived cytokines such as IL-12 and TNF- � have been shown to play a critical role in the de-struction of pancreatic beta cells in a rat model of insulin-dependent diabetes mellitus [23] . Also, it has been report-ed that up-regulation of iNOS in macrophages promoted islet beta cell destruction in the NOD mouse [24] . Simi-larly, iNOS, TNF- � and IL-12 from macrophages have been demonstrated to be important factors causing renal inflammation and fibrosis in many type of chronic kid-ney diseases [25–27] . In this study, we demonstrated in vitro that IL-12, TNF- � and iNOS were up-regulated in macrophages by glucose in high concentration, which is line with the finding of Wen et al. [28] . Importantly, M2 macrophages were shown to deactivate macrophages ex-posed to high glucose by reducing the expression of iNOS and inflammatory cytokines in vitro. It is the first dem-onstration of the ability of M2 macrophages to deactivate macrophages that have been activated specifically by high glucose. This deactivating capacity of M2 macro-

phages was further demonstrated in vivo, as a reduction in iNOS and inflammatory cytokine expression of en-dogenous renal macrophages. This could be one of the mechanisms underlying the protective effect of M2 mac-rophages against renal injury.

In this study, the protection of renal structure was not accompanied by renal functional protection. This is not surprising as during its early stages renal function in mu-rine STZ-induced diabetes remains largely unchanged. Moreover, it has been reported that there is a poor or no relationship between renal function and renal structure in various diabetic models [29, 30] . It is possible that ef-fects of M2 macrophages on renal function could become apparent later in the course of disease or in models with more severe renal dysfunction.

In type I diabetes, the infiltration of islets with macro-phages precedes and drives lymphocytic insulitis and mediates islet injury by oxidants and cytokines [31–33] . Activated CD4+ and CD8+ T cells contribute to the pathogenesis of type 1 diabetes by secreting IFN- � and destroying beta cells. Suppression or inhibition of macro-phages and their products, such as nitric oxide, was dem-onstrated to reduce the severity of insulitis in NOD mice [34] and to prevent streptozotocin-induced diabetes [35–37] . Knockout of Uncoupling protein 2, a mitochondrial protein which controls macrophage activation, increased macrophage recruitment into islets of STZ-treated mice

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Fig. 6. Deactivation of endogenous kidney macrophages (EM) by M2 macrophages. a EM (CD11b+DiI–) were separated from STZ mice transfused with M2 macrophages by FACS sorting at day 70. b EM were isolated from kidney from each group at day 70, mRNA expression of cytokines was examined by real-time PCR. * p ! 0.01 vs. diabetic control and diabetic-M0. Data represent the mean 8 SD, n = 7 per group.



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and exacerbated disease [38] . Similarly, suppression of ef-fector T cells by regulatory T cells was shown to reverse diabetes in autoimmune NOD mice [39] . In the current study, large pancreatic infiltrates of macrophages and T cells were found in diabetic controls during the first 2 weeks, whereas the number of inflammatory cells was reduced greatly in diabetic mice transfused with M2 macrophages.

It is conceivable that the protective effect of M2 mac-rophages against renal injury in diabetes could involve reduced destruction of insulin secreting cells in pancreas, and consequent reduced severity of diabetes. However, there was no correlation in diabetic mice transfused with M2 macrophages between the severity of functional and structural renal injury and functional and structural islet injury. It could have been due to both indirect and direct effects of M2 on renal injury in diabetic nephropathy. Ad-ministration of M2 macrophages protected against pan-creas injury and improved the diabetic state and thus could have consequently reduced renal inflammation

and fibrosis. These findings could be a direct contribu-tion of the M2 macrophages to reduction of inflamma-tion and lessening of renal injury.

In conclusion, our study shows that alternatively acti-vated macrophages can effectively reduce the severity of both experimental diabetes and diabetic nephropathy.

Disclosure Statement

The authors have no financial conflict of interest.

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia (NHMRC, grant 457345 to Dr. Y. Wang), and Johnson and Johnson Research Pty Ltd. (focused funding to Prof. D. Harris). The authors acknowledge Dr. Karen Byth for help with statistical analysis and Ms. Aysen Yuksel of Westmead Millennium Institute for assistance with histological examination.



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transfused macrophages ameliorate pancreatic and renal injury in murine diabetes mellitus

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