- Email: [email protected]
Macrophages, Dendritic Cells, and Kidney Ischemia-Reperfusion Injury
Macrophages, Dendritic Cells, and Kidney Ischemia-Reperfusion Injury Li Li, MD, PhD, and Mark D. Okusa, MD Summary: Dendritic cells and macrophages are critical early initiators of innate immunity in the kidney and orchestrate inflammation subsequent to ischemia-reperfusion injury. They are the most abundant leukocytes present in the kidney, and they represent a heterogeneous population of cells that are capable of inducing sterile inflammation after reperfusion directly through the production of proinflammatory cytokines and other soluble inflammatory mediators or indirectly through activation of effector T lymphocytes and natural killer T cells. In addition, recent studies have indicated that kidney and immune cell micro-RNAs control gene expression and have the ability to regulate the initial inflammatory response to injury. Although dendritic cells and macrophages contribute to both innate and adaptive immunity and to injury and repair, this review focuses on the initial innate response to kidney ischemia-reperfusion injury. Semin Nephrol 30:268-277 © 2010 Elsevier Inc. All rights reserved. Keywords: Antigen presentation, leukocyte, innate immunity, inflammation
endritic cells and macrophages play an important role in the innate and adaptive immune response of acute ischemia-reperfusion injury (IRI). In the kidney they reside in the interstitial extracellular compartment and are poised to interact with substances transported from the tubule lumen into peritubular capillaries,1 endogenous molecules released from parenchymal cells or exogenous invading organisms, or with resident or infiltrating immune cells including lymphocytes, natural killer T (NKT) cells, epithelial cells, and fibroblasts. Electron microscopy has identified characteristics that may differentiate dendritic cells (DCs) from macrophages. Dendritic cells are characterized by Birbeck granules, specialized compartments serving as reservoirs for antigen processing, and they have a greater density of mitochondria and rough endoplasmic reticulum, larger Golgi, and fewer lysosomes1
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Department of Medicine and the Center for Immunity, Inflammation and Regenerative Medicine, University of Virginia, Charlottesville, VA. Supported in part by funds from the National Institutes of Health (RO1DK56223 and RO1DK62324), Genzyme (Genzyme Renal Innovations Program), and an American Heart Associate National Scientist Development grant (0835258N). Address reprint requests to Mark D. Okusa, MD, Division of Nephrology, Box 800133, University of Virginia Health System, Charlottesville, VA 22908. E-mail: [email protected] 0270-9295/ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2010.03.005
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than macrophages. Dendritic cells and macrophages are key initiators, potentiators, and effectors of innate immunity in kidney IRI and induce injury either through inflammatory signals to other effector cells or directly through the release of soluble mediators. The early immune response consists of activation of DCs and macrophages that produce cytokines and chemokines, leading to a prompt influx of leukocytes. In addition, DCs, potent antigen-presenting cells, contribute to the innate immune response by activating NKT cells and promoting inflammation. After activation, macrophage and DC subpopulations subsequently contribute to the resolution of injury. Although often discussed as discrete cells, the heterogeneous population of antigen-presenting cells represents a continuum from macrophages to DCs2 that contribute to both the early and late phases of kidney IRI3,4 (Fig. 1). Although the role of antigen-presenting cells in the resolution of injury are described elsewhere, this article focuses on the early initiating events of kidney IRI for which macrophages and DCs serve as catalysts for subsequent inflammation. OVERVIEW OF ACUTE KIDNEY IRI The initial event leading to tissue injury in kidney IRI is the acute reduction of blood flow that
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Figure 1. Heterogeneity of antigen-presenting cells in the kidney. Kidney section of the outer medulla from a C57BL/6 background CX3CR1⫹/GFP mouse. GFP is expressed mainly on monocyte/macrophages and DCs (green). Sections also were stained for MCH class II (PE-tagged IA-positive cells). High magnification of images of the kidney medulla viewed under a Zeiss LSM-510 confocal microscope showed CX3CR1-GFP⫹ cells in green and PE-tagged IA⫹ cells in red, identifying DCs. Scale bar, 50 m.
produces hypoxia-induced vascular and tubular dysfunction. The S3 segment of the proximal tubule is located in the renal medulla and is particularly susceptible to injury in part because of the normally low ambient oxygen tension (PO2, 5-20 mm Hg).5,6 After reperfusion, various intracellular events occur that lead to cellular dysfunction, apoptosis, and cell death (for reviews see Thadhani et al,7 Bonventre and Weinberg,8 and Schrier et al9). Mitochondria become swollen and fragmented with reduced membrane potential and generate reactive oxygen species and nitric oxide.10,11 Intracellular calcium accumulates12,13 and membranes and lipids are damaged,14,15 whereas nuclear factor-B signaling pathways16 and adenosine diphosphate ribose (ADP) polymerase are activated.17 Subsequent alterations in ultrafiltration coefficient, tubular obstruction, and/or back-leak reduce the glomerular filtration rate, resulting in acute renal dysfunction. The innate immune system, leading to the activation of bone marrow– derived cells,
endothelial cells, and epithelial cells, significantly contributes to kidney IRI.3,18 INFLAMMATION IN ACUTE KIDNEY IRI Inflammation is an important early event leading to intracellular responses that ultimately result in apoptosis and necrosis. Breakdown of the regulation of inflammatory responses results in tissue injury despite the concurrent activation of alternative pathways that eventually lead to remodeling and tissue repair. Thus, the involvement of the immune system in kidney IRI is very complex. Delineation of the orchestration of immune cells after kidney IRI has been made possible with the application of flow cytometry and the use of genetically modified mice, before which quantitative analysis relied primarily on immunohistochemical labeling of small samples of tissue. By using multicolor flow cytometry, a comprehensive profile of resident and infiltrating leukocytes after kid-
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Figure 2. Flow cytometric analysis of intrarenal leukocyte populations in kidney IRI. Suspended kidney cells were stained and, by gating on the CD45⫹7AAD⫺ live leukocyte population, F4/80⫹ macrophages were detected by flow cytometry. The profile of macrophage influx into kidneys in comparison with other leukocytes (as determined by flow cytometric analysis) after IRI is shown in the early (0.5-24 h) and late (48-148 h) phases of reperfusion. GR-1, neutrophils; B220, B cells; CD4 and CD8, T cells; F4/80, macrophages.
ney IRI has been described.19 Resident kidney DCs are the dominant leukocyte subset and are distributed throughout the whole kidney.19 Figure 2 illustrates the temporal profile of leukocyte trafficking after kidney IRI. The appearance in the kidneys of neutrophils and macrophages— components of the innate immune system—within 30 minutes after reperfusion, provides support for their pathogenic role in kidney IRI.19-21 CD4⫹ and CD8⫹ T cells and B220⫹ cells (B cells) also rapidly infiltrate the kidney within 30 minutes after reperfusion. Both neutrophil and macrophage infiltration peaks at 24 to 48 hours and remains increased for at least 6 days after reperfusion injury. Although effective in eradicating pathogens, a consequence of such a stereotypical, generalized response is secondary tissue injury. Den-
dritic cells, neutrophils, phagocytic macrophages, and lymphocytes participate in the early phase of kidney IRI as well as the late reparative phase. By using flow cytometry and immunofluorescence microscopy the kinetics of neutrophil migration from intravascular to interstitial compartments has been shown clearly.22 In these studies neutrophils marginate and then transmigrate across the vascular wall into the surrounding interstitium where they damage tissue. The early activation of macrophages and DCs leads to the infiltration of neutrophils with DC generation of interleukin (IL)-23 contributing to the activation of neutrophils and leading to the production of the proinflammatory cytokine IL-17.23 Thus, accumulating data suggest that DCs and macrophages contribute early in the innate immune
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response to kidney IRI and promote neutrophil infiltration. However, although DCs and macrophages contribute to kidney IRI, T-regulatory cells have been shown to suppress the extent of kidney IRI through an IL-10 – dependent mechanism.24 The innate immune system has evolved as a host defense mechanism that recognizes microbial products. Toll-like receptors (TLRs) and a limited number of other receptors respond to highly conserved structures referred to as pathogen-associated molecular patterns, leading to a rapid and stereotypical response.25,26 Although this concept describes the initiation of inflammation after infections, similar processes that initiate inflammation are likely to occur in response to tissue trauma and autoimmune inflammation.27 A number of molecular patterns associated with release of endogenous cellular molecules, such as mammalian DNA, RNA, heat shock proteins, interferon-␣, IL-1, CD40-L, fibronectin fragments, modified lowdensity lipoproteins, extracellular adenosine triphosphate, and high-mobility group box chromosomal protein 1, may initiate the inflammatory responses of the TLR and nucleotide-binding oligomerization domain (NOD)like receptor family of receptors.28 Danger signals that permit rapid cellular communication are a key conceptual component of immune activation in both infection and sterile tissue injury. Activation of TLRs results in rapid changes in the expression of genes encoding cytokines, degradative enzymes, and enzymes involved in the production of multiple low molecular weight inflammatory mediators.29 Once activated, an inflammatory and immune response leads to sequestration of leukocytes in inflamed sites, complement activation, and eradication of pathogens through cytokines, complement/membrane attack complex, and the actions of NKT cells.3,4,18 HETEROGENEITY OF KIDNEY MACROPHAGES AND DENDRITIC CELLS Blood monocytes are a heterogeneous cell population and are the precursors of tissue macrophages and DCs (Fig. 3). At least 2 distinct blood monocyte subsets are found in mice depending
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on the time they spend in the bloodstream.30-33 Monocyte subsets migrate into normal tissue and differentiate into resident DCs and macrophages,32,34 and they infiltrate inflamed tissue and differentiate into activated macrophages or DCs.31 Resident monocytes are characterized as CCR2⫺CX3CR1highGR-1⫺Ly6C⫺, whereas inflammatory monocytes are defined as CCR2 ⫹ CX3CR1lowGR-1⫹Ly6Chigh.31 After tissue damage or infection, a coordinated multistep response is thought to occur involving the circulating resident monocyte population that expresses LFA-1 and CX3CR1. These resident monocytes patrol and monitor the endothelium of normal tissue and, after injury, rapidly migrate into the inflamed tissue where they differentiate into macrophages and initiate an early innate immune response.34 The prevailing microenvironment within the tissue plays a key role in determining macrophage phenotype. Tumor necrosis factor (TNF)-␣, IL-4, and IL-15 skew monocyte differentiation toward DCs35-37 whereas interferon (IFN)-␥ and IL-6 direct monocyte differentiation toward macrophages.38,39 The macrophage phenotype switch occurs over time and parallels the course of inflammation or infection. Thus, it is likely that the phenotype of macrophages and DCs in inflamed tissues is the result of transmigration of cells derived from subsets of circulating monocytes as well as phenotypic changes of macrophages and DCs made in response to cues within the local microenvironment. MACROPHAGE AND DENDRITIC CELL TRAFFICKING IN KIDNEY ISCHEMIA-REPERFUSION Given their diverse functions, macrophages might participate in early stages of injury or in the late-stage repair process after kidney IRI. Monocytes/macrophages appear in the kidney within 1 to 5 days of IRI,40-43 as do macrophageassociated cytokines (eg, IL-1, IL-6, and transforming growth factor-)41 and the monocyte/ macrophage chemoattractants IFN-␥–inducible 10-kilodalton protein, monocyte chemotactic protein-1, and macrophage inflammatory protein 2.44-46 Monocyte/macrophage migration into inflamed atherosclerotic vessels depends
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Figure 3. Trafficking of monocytes in mice after kidney IRI. Bone marrow CD11b⫹Ly6Chigh monocyte/monocyte precursor egress to the blood circulation is CCR2-dependent. (a) Some of the Ly6Chigh monocytes lose their CCR2 and Ly6C expression and are characterized further as being CD62L⫺GR-1⫺CX3CR1high. (b) These resident monocytes migrate to normal noninflamed tissue rapidly after they are released in the blood and differentiate into tissue DCs, which are CD11b⫹CD11c⫹IAhigh CD86⫹F4/80high. (c) On the other hand, some of the monocytes continue to express CCR2⫹ and Ly6Chigh on the cell surface and also are CX3CR1lowGR-1intCD62L⫹. (d) These inflammatory monocytes respond to the gradient of chemokines (e) released from kidneys after IRI. In the injured tissue, the macrophages derived from inflamed monocytes are characterized as being CD62L⫹Gr-1intLy6C⫹F4/80low. Infiltrating macrophages produce large amounts of proinflammatory cytokines, which are involved in tissue injury.
on CCR2, CCR5, and CX3CR1,47 and similarly migration of monocyte/macrophages in kidney IRI depends on CX3CR119,48 and CCR2.19 CCR2deficient mice were protected from kidney IRI and this protection was associated with reduced macrophage infiltration.49 A causal relationship between CCR2⫹ monocyte/macrophages and tissue injury after kidney IRI was shown by protection from injury and reduced monocyte infiltration into injured tissue after adoptive transfer of CCR2⫺/⫺ monocytes to bone marrow–ablated mice. In contrast, reconstitution with CCR2⫹/⫹ monocytes facilitated
kidney macrophage infiltration and induced tissue injury. Therefore, inflammatory monocyte migration into the reperfused kidney is, at least in part, CCR2-dependent and contributes to the pathogenesis of early inflammation in kidney IRI. Fractalkine (CX3CL1) is a macrophage chemoattractant expressed on the surface of endothelial cells and is the ligand for the fractalkine receptor (CX3CR1). CX3CR1lowGR-1⫹CCR2⫹ subsets of monocytes actively are recruited to inflamed tissue whereas CX3CR1highGR-1⫺CCR2⫺ subsets of monocytes migrate to uninjured tissue
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and differentiate into resident macrophages and DCs.33 CX3CR1 deficiency (CX3CR1GFP/GFP mice; GFP, green fluorescent protein) or treatment with a CX3CR1-blocking antibody48 protected kidneys from IRI, thereby indicating that CX3CR1 is a key signal that mediates infiltration of inflammatory monocytes into injured kidneys after IRI. FUNCTIONAL ROLE OF MACROPHAGES AND DENDRITIC CELLS IN IRI Classic depletion and reconstitution studies have established the role of macrophages and DCs in renal injury.50,51 For example, nitrogen mustard or anti-macrophage serum was used to abrogate injury in a model of glomerulonephritis with the protective effect being lost after the reconstitution of macrophages.51 Macrophage depletion using bisphosphonate clodronate (dichloromethylene bisphosphonate) protected tissue from IRI,43,52 providing strong evidence for the participation of macrophages in the induction of injury after IRI. The role of DCs can be assessed more specifically through the use of transgenic mice expressing the human diphtheria toxin receptor (DTR; human heparin binding epidermal growth factor-like growth factor) in CD11c⫹ cells (CD11c-DTR transgenic mouse).53 Transgenic expression of the human DTR renders normally DT-resistant murine cells DT sensitive. By using this inducible lineage ablation method, exposure of CD11c-DTR mice to low-dose DT will kill primarily CD11c⫹ DCs. Kidney CD11c⫹GFP⫹ DCs were depleted efficiently in CD11c-DTR/GFP mice by DT but not by mutant DT, as revealed by immunofluorescence studies (Fig. 4A). Kidney injury, as indicated by an increase in plasma creatinine level (Fig. 4B) or H&E staining showing tubular cell necrosis (Fig. 4C), was significantly less in the CD11c-DTR/GFP mice that received DT treatment before IRI in comparison with control mutant DT treatment. The protective effect was not evident in DT- or mutant DT-treated wildtype control mice (Fig. 4B and C). Thus, DCs actively contribute to the early innate injurious response in kidney IRI.
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EFFECTOR MECHANISMS OF ANTIGEN-PRESENTING CELLS IN KIDNEY IRI
Activation of NKT Cells in Kidney IRI Although CD4⫹ cells are known to contribute to kidney IRI, the antigen-specific activation of T cells typically contributes to adaptive immunity and is a process not consistent with acute IRI that occurs within 24 hours. However, CD4⫹ T cells consist of functionally distinct subsets, including NKT cells that participate in innate immunity and could contribute to kidney IRI. CD4⫹ cells were found to infiltrate the kidney within 30 minutes after IRI (Fig. 2), and IFN-␥–producing CD1d-restricted NKT cells were identified early in kidney IRI. Furthermore, significant protection from kidney IRI was evident in mice administered a CD1d monoclonal antibody that blocks the interaction between antigen-presenting cells and NKT cells as well as mice deficient in NKT cells (Ja18⫺/⫺).54 These studies suggest that the CD1d molecule expressed on kidney DCs can present endogenous glycolipid to NKT cells (signal 1), thereby stimulating CD40-CD40L interaction (signal 2) and cytokine (IL-12) production (signal 3) to promote the innate immune response.55 Activation of CD1d-restricted NKT cells also promotes IFN-␥–producing GR-1⫹ neutrophil infiltration and tissue inflammation after kidney IRI.54
Soluble Mediators and Phagocytosis in Inflammation Macrophages and DCs are involved in antigen presentation and immunoregulation, and they express a large number of cell-surface proteins that play an important role in the phagocytic function of these cells. Activation of macrophages and DCs produces proinflammatory cytokines such as IL-1␣, IL-6, IL-12, IL-18, and TNF-␣ via MyD-88 – dependent or MyD-88 –independent pathways. The observation that kidneys produce IL-1 and IL-18 after IRI56 suggests the potential involvement of the inflammasome, a multiprotein complex in macrophages and DCs, in the inflammatory processes.57 NOD-like receptors possess pattern recognition receptors and are important sensors of microbial products and other pathogen-associ-
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Figure 4. DCs are important in the pathogenesis of kidney IRI. (A) CD11c⫹GFP⫹ cells (arrows) in mouse kidney sections from CD11c-DTR/GFP mice (which express the human DTR as a GFP fusion protein) were revealed immunohistochemically with GFP antibody (fluorescein isothiocyanate fluorescence, green). Biologically inactive mutant DT had no effect on the number of GFP-positive CD11c cells in the CD11c-DTR/GFP sham and IRI mouse kidneys. However, there were significantly less GFP-labeled cells in the sham and IRI kidneys of CD11c-DTR/GFP mice after administration of bioactive DT. Scale bar, 50 m. (B) Plasma creatinine level was measured in wild-type (WT) and CD11c-DTR/GFP mice that received mutant (control) or bioactive DT (4 g/g) 48 hours before surgery. Values are mean ⫾ SE; N ⫽ 2-11. ***P ⬍ .001. (C) H&E staining of kidneys from WT and CD11c-DTR/GFP mice reveals marked tubular injury in WT kidneys after IRI. Pretreatment with bioactive but not mutant DT before kidney IRI protected CD11c-DTR mouse kidneys from injury. Arrows indicate necrotic tubules. Scale bar, 100 m. (A and C) Data are representative of more than 3 experiments. Data from Li and Okusa, unpublished data, 2010.
ated molecular patterns. Once NOD-like receptors recognize microbial products or endogenous danger signals, this leads to activation of caspase 1 and assembly of the inflammasome. Caspase 1 then promotes the maturation of the inflammatory cytokines IL-1 and IL-18.58 No studies to date have examined the role of inflammasomes in acute kidney IRI. Insights into the mechanisms by which macrophages induce tissue injury may be gained from studies of their role in infection. The an-
timicrobial action of macrophages is mediated through oxidative mechanisms including the production of reactive oxygen intermediates by phagocyte oxidase and reactive nitrogen intermediates generated by inducible nitric oxide synthase 2(iNOS2).59,60 Macrophages also exert antimicrobial actions via a phagocyte oxidase– and nitric oxide synthase 2–independent mechanism.61 In addition, nonoxidative antimicrobial actions by macrophages may induce tissue injury. Macrophage-specific metalloelastase is in-
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volved in remodeling of the extracellular matrix associated with inflammation and the repair process.62 Macrophages also generate matrix metalloproteinase 12, a powerful enzyme that adheres to the bacterial wall to mediate bacterial killing but also degrades matrix proteins during inflammation. Thus, propensity of macrophages to elaborate these effective mediators for bacterial killing also may lead to tissue injury after the sterile inflammatory response that follows kidney IRI.
Macrophage and Dendritic Cell MicroRNAs in Inflammation MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by either degradation or translational repression of a target messenger RNA. They play a critical role in gene regulation through their stability and translation in the kidney.63,64 For example, uremia associated with acute kidney injury suppressed proinflammatory cytokine messenger RNA levels and altered the expression of at least 69 miRNAs.65 A number of miRNAs contribute to the maturation of macrophages via TLR signaling. TLR and IFN- stimulate production of miRNAs by the nuclear factor-B and Jun N-terminal kinase pathway. TNF-␣ is a target gene of miR-125b and down-regulation of miRNA-125b is required to ensure that a proper inflammatory response is generated by macrophages in response to microbial stimulation.66 The miRNA-146a reduces the expression of IRAK1 and TRAF6 of the TLR signaling cascade and thus acts to provide negative feedback to prevent excessive inflammation.67 Several TLR ligands increase miR-155 expression via the MyD88 or TRIF signal pathway and miR-155 is up-regulated by lipopolysaccharide and promotes TNF-␣ production from macrophages.68 Furthermore, miR-155 also regulates DC antigen presentation and costimulation activity with DCs from miR-155 knockout mice is unable to induce efficient T-cell activation.67 These studies show that a variety of miRNAs act to regulate DC and macrophage function and thus may participate in modulating tissue injury after kidney IRI.
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CONCLUSIONS DCs and macrophages are the most abundant leukocytes expressed in the normal kidney. They represent a continuum of cell phenotype in which macrophages, which have poor antigen processing and presentation properties, produce proinflammatory cytokines and soluble mediators that contribute to innate immunity in kidney IRI. At the other end of the spectrum are DCs, which, on activation, function in processing and presenting antigen to effector T cells. Presentation of endogenous glycolipid to NKT cells after kidney IRI initiates injurious responses. The early activation of DCs and macrophages orchestrates an immune response that leads to sterile inflammation and associated tissue damage and these immune cells likely will serve as important therapeutic targets in the future. ACKNOWLEDGMENTS The authors acknowledge Ms. Liping Huang and Ms. Hong Ye (Department of Medicine, University of Virginia) for generation of much of the data for this article; and Dr. Diane Rosin (Department of Pharmacology, University of Virginia) for helpful discussions and careful reading of the manuscript.
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et al. NKT cell activation mediates neutrophil IFNgamma production and renal ischemiareperfusion injury. J Immunol. 2007;178:589-911. Hayakawa Y, Takeda K, Yagita H, Van Kaer L, Saiki I, Okumura K. Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways. J Immunol. 2001;166:6012-8. Melnikov VY, Ecder T, Fantuzzi G, Siegmund B, Lucia MS, Dinarello CA, et al. Impaired IL18 processing protects caspase1deficient mice from ischemic acute renal failure. J Clin Invest. 2001;107:1145-52. Mariathasan S, Monack DM. Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol. 2007;7:31-40. Ogura Y, Sutterwala FS, Flavell RA. The inflammasome: first line of the immune response to cell stress. Cell. 2006;126:659-62. Nathan CF, Hibbs JB Jr. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol. 1991;3:65-70. Nathan CF. Mechanisms of macrophage antimicrobial activity. Trans R Soc Trop Med Hyg. 1983;77:620-30. Shiloh MU, MacMicking JD, Nicholson S, Brause JE, Potter S, Marino M, et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity. 1999; 10:29-38. Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD. Metalloelastase is required for macrophagemediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci U S A. 1996;93:3942-6. Houghton AM, Hartzell WO, Robbins CS, GomisRuth FX, Shapiro SD. Macrophage elastase kills bacteria within murine macrophages. Nature. 2009;460:637-41. Saal S, Harvey SJ. MicroRNAs and the kidney: coming of age. Curr Opin Nephrol Hypertens. 2009;18:317-23. Zager RA, Johnson AC, Lund S. Uremia impacts renal inflammatory cytokine gene expression in the setting of experimental acute kidney injury. Am J Physiol Renal Physiol. 2009;297:F961-70. Bi Y, Liu G, Yang R. MicroRNAs: novel regulators during the immune response. J Cell Physiol. 2009; 218:467-72. Lu LF, Liston A. MicroRNA in the immune system, microRNA as an immune system. Immunology. 2009; 127:291-8. O’Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. MicroRNA155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci U S A. 2007;104:1604-9.
endritic cells and macrophages play an important role in the innate and adaptive immune response of acute ischemia-reperfusion injury (IRI). In the kidney they reside in the interstitial extracellular compartment and are poised to interact with substances transported from the tubule lumen into peritubular capillaries,1 endogenous molecules released from parenchymal cells or exogenous invading organisms, or with resident or infiltrating immune cells including lymphocytes, natural killer T (NKT) cells, epithelial cells, and fibroblasts. Electron microscopy has identified characteristics that may differentiate dendritic cells (DCs) from macrophages. Dendritic cells are characterized by Birbeck granules, specialized compartments serving as reservoirs for antigen processing, and they have a greater density of mitochondria and rough endoplasmic reticulum, larger Golgi, and fewer lysosomes1
D
Department of Medicine and the Center for Immunity, Inflammation and Regenerative Medicine, University of Virginia, Charlottesville, VA. Supported in part by funds from the National Institutes of Health (RO1DK56223 and RO1DK62324), Genzyme (Genzyme Renal Innovations Program), and an American Heart Associate National Scientist Development grant (0835258N). Address reprint requests to Mark D. Okusa, MD, Division of Nephrology, Box 800133, University of Virginia Health System, Charlottesville, VA 22908. E-mail: [email protected] 0270-9295/ - see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2010.03.005
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than macrophages. Dendritic cells and macrophages are key initiators, potentiators, and effectors of innate immunity in kidney IRI and induce injury either through inflammatory signals to other effector cells or directly through the release of soluble mediators. The early immune response consists of activation of DCs and macrophages that produce cytokines and chemokines, leading to a prompt influx of leukocytes. In addition, DCs, potent antigen-presenting cells, contribute to the innate immune response by activating NKT cells and promoting inflammation. After activation, macrophage and DC subpopulations subsequently contribute to the resolution of injury. Although often discussed as discrete cells, the heterogeneous population of antigen-presenting cells represents a continuum from macrophages to DCs2 that contribute to both the early and late phases of kidney IRI3,4 (Fig. 1). Although the role of antigen-presenting cells in the resolution of injury are described elsewhere, this article focuses on the early initiating events of kidney IRI for which macrophages and DCs serve as catalysts for subsequent inflammation. OVERVIEW OF ACUTE KIDNEY IRI The initial event leading to tissue injury in kidney IRI is the acute reduction of blood flow that
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Figure 1. Heterogeneity of antigen-presenting cells in the kidney. Kidney section of the outer medulla from a C57BL/6 background CX3CR1⫹/GFP mouse. GFP is expressed mainly on monocyte/macrophages and DCs (green). Sections also were stained for MCH class II (PE-tagged IA-positive cells). High magnification of images of the kidney medulla viewed under a Zeiss LSM-510 confocal microscope showed CX3CR1-GFP⫹ cells in green and PE-tagged IA⫹ cells in red, identifying DCs. Scale bar, 50 m.
produces hypoxia-induced vascular and tubular dysfunction. The S3 segment of the proximal tubule is located in the renal medulla and is particularly susceptible to injury in part because of the normally low ambient oxygen tension (PO2, 5-20 mm Hg).5,6 After reperfusion, various intracellular events occur that lead to cellular dysfunction, apoptosis, and cell death (for reviews see Thadhani et al,7 Bonventre and Weinberg,8 and Schrier et al9). Mitochondria become swollen and fragmented with reduced membrane potential and generate reactive oxygen species and nitric oxide.10,11 Intracellular calcium accumulates12,13 and membranes and lipids are damaged,14,15 whereas nuclear factor-B signaling pathways16 and adenosine diphosphate ribose (ADP) polymerase are activated.17 Subsequent alterations in ultrafiltration coefficient, tubular obstruction, and/or back-leak reduce the glomerular filtration rate, resulting in acute renal dysfunction. The innate immune system, leading to the activation of bone marrow– derived cells,
endothelial cells, and epithelial cells, significantly contributes to kidney IRI.3,18 INFLAMMATION IN ACUTE KIDNEY IRI Inflammation is an important early event leading to intracellular responses that ultimately result in apoptosis and necrosis. Breakdown of the regulation of inflammatory responses results in tissue injury despite the concurrent activation of alternative pathways that eventually lead to remodeling and tissue repair. Thus, the involvement of the immune system in kidney IRI is very complex. Delineation of the orchestration of immune cells after kidney IRI has been made possible with the application of flow cytometry and the use of genetically modified mice, before which quantitative analysis relied primarily on immunohistochemical labeling of small samples of tissue. By using multicolor flow cytometry, a comprehensive profile of resident and infiltrating leukocytes after kid-
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Figure 2. Flow cytometric analysis of intrarenal leukocyte populations in kidney IRI. Suspended kidney cells were stained and, by gating on the CD45⫹7AAD⫺ live leukocyte population, F4/80⫹ macrophages were detected by flow cytometry. The profile of macrophage influx into kidneys in comparison with other leukocytes (as determined by flow cytometric analysis) after IRI is shown in the early (0.5-24 h) and late (48-148 h) phases of reperfusion. GR-1, neutrophils; B220, B cells; CD4 and CD8, T cells; F4/80, macrophages.
ney IRI has been described.19 Resident kidney DCs are the dominant leukocyte subset and are distributed throughout the whole kidney.19 Figure 2 illustrates the temporal profile of leukocyte trafficking after kidney IRI. The appearance in the kidneys of neutrophils and macrophages— components of the innate immune system—within 30 minutes after reperfusion, provides support for their pathogenic role in kidney IRI.19-21 CD4⫹ and CD8⫹ T cells and B220⫹ cells (B cells) also rapidly infiltrate the kidney within 30 minutes after reperfusion. Both neutrophil and macrophage infiltration peaks at 24 to 48 hours and remains increased for at least 6 days after reperfusion injury. Although effective in eradicating pathogens, a consequence of such a stereotypical, generalized response is secondary tissue injury. Den-
dritic cells, neutrophils, phagocytic macrophages, and lymphocytes participate in the early phase of kidney IRI as well as the late reparative phase. By using flow cytometry and immunofluorescence microscopy the kinetics of neutrophil migration from intravascular to interstitial compartments has been shown clearly.22 In these studies neutrophils marginate and then transmigrate across the vascular wall into the surrounding interstitium where they damage tissue. The early activation of macrophages and DCs leads to the infiltration of neutrophils with DC generation of interleukin (IL)-23 contributing to the activation of neutrophils and leading to the production of the proinflammatory cytokine IL-17.23 Thus, accumulating data suggest that DCs and macrophages contribute early in the innate immune
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response to kidney IRI and promote neutrophil infiltration. However, although DCs and macrophages contribute to kidney IRI, T-regulatory cells have been shown to suppress the extent of kidney IRI through an IL-10 – dependent mechanism.24 The innate immune system has evolved as a host defense mechanism that recognizes microbial products. Toll-like receptors (TLRs) and a limited number of other receptors respond to highly conserved structures referred to as pathogen-associated molecular patterns, leading to a rapid and stereotypical response.25,26 Although this concept describes the initiation of inflammation after infections, similar processes that initiate inflammation are likely to occur in response to tissue trauma and autoimmune inflammation.27 A number of molecular patterns associated with release of endogenous cellular molecules, such as mammalian DNA, RNA, heat shock proteins, interferon-␣, IL-1, CD40-L, fibronectin fragments, modified lowdensity lipoproteins, extracellular adenosine triphosphate, and high-mobility group box chromosomal protein 1, may initiate the inflammatory responses of the TLR and nucleotide-binding oligomerization domain (NOD)like receptor family of receptors.28 Danger signals that permit rapid cellular communication are a key conceptual component of immune activation in both infection and sterile tissue injury. Activation of TLRs results in rapid changes in the expression of genes encoding cytokines, degradative enzymes, and enzymes involved in the production of multiple low molecular weight inflammatory mediators.29 Once activated, an inflammatory and immune response leads to sequestration of leukocytes in inflamed sites, complement activation, and eradication of pathogens through cytokines, complement/membrane attack complex, and the actions of NKT cells.3,4,18 HETEROGENEITY OF KIDNEY MACROPHAGES AND DENDRITIC CELLS Blood monocytes are a heterogeneous cell population and are the precursors of tissue macrophages and DCs (Fig. 3). At least 2 distinct blood monocyte subsets are found in mice depending
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on the time they spend in the bloodstream.30-33 Monocyte subsets migrate into normal tissue and differentiate into resident DCs and macrophages,32,34 and they infiltrate inflamed tissue and differentiate into activated macrophages or DCs.31 Resident monocytes are characterized as CCR2⫺CX3CR1highGR-1⫺Ly6C⫺, whereas inflammatory monocytes are defined as CCR2 ⫹ CX3CR1lowGR-1⫹Ly6Chigh.31 After tissue damage or infection, a coordinated multistep response is thought to occur involving the circulating resident monocyte population that expresses LFA-1 and CX3CR1. These resident monocytes patrol and monitor the endothelium of normal tissue and, after injury, rapidly migrate into the inflamed tissue where they differentiate into macrophages and initiate an early innate immune response.34 The prevailing microenvironment within the tissue plays a key role in determining macrophage phenotype. Tumor necrosis factor (TNF)-␣, IL-4, and IL-15 skew monocyte differentiation toward DCs35-37 whereas interferon (IFN)-␥ and IL-6 direct monocyte differentiation toward macrophages.38,39 The macrophage phenotype switch occurs over time and parallels the course of inflammation or infection. Thus, it is likely that the phenotype of macrophages and DCs in inflamed tissues is the result of transmigration of cells derived from subsets of circulating monocytes as well as phenotypic changes of macrophages and DCs made in response to cues within the local microenvironment. MACROPHAGE AND DENDRITIC CELL TRAFFICKING IN KIDNEY ISCHEMIA-REPERFUSION Given their diverse functions, macrophages might participate in early stages of injury or in the late-stage repair process after kidney IRI. Monocytes/macrophages appear in the kidney within 1 to 5 days of IRI,40-43 as do macrophageassociated cytokines (eg, IL-1, IL-6, and transforming growth factor-)41 and the monocyte/ macrophage chemoattractants IFN-␥–inducible 10-kilodalton protein, monocyte chemotactic protein-1, and macrophage inflammatory protein 2.44-46 Monocyte/macrophage migration into inflamed atherosclerotic vessels depends
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Figure 3. Trafficking of monocytes in mice after kidney IRI. Bone marrow CD11b⫹Ly6Chigh monocyte/monocyte precursor egress to the blood circulation is CCR2-dependent. (a) Some of the Ly6Chigh monocytes lose their CCR2 and Ly6C expression and are characterized further as being CD62L⫺GR-1⫺CX3CR1high. (b) These resident monocytes migrate to normal noninflamed tissue rapidly after they are released in the blood and differentiate into tissue DCs, which are CD11b⫹CD11c⫹IAhigh CD86⫹F4/80high. (c) On the other hand, some of the monocytes continue to express CCR2⫹ and Ly6Chigh on the cell surface and also are CX3CR1lowGR-1intCD62L⫹. (d) These inflammatory monocytes respond to the gradient of chemokines (e) released from kidneys after IRI. In the injured tissue, the macrophages derived from inflamed monocytes are characterized as being CD62L⫹Gr-1intLy6C⫹F4/80low. Infiltrating macrophages produce large amounts of proinflammatory cytokines, which are involved in tissue injury.
on CCR2, CCR5, and CX3CR1,47 and similarly migration of monocyte/macrophages in kidney IRI depends on CX3CR119,48 and CCR2.19 CCR2deficient mice were protected from kidney IRI and this protection was associated with reduced macrophage infiltration.49 A causal relationship between CCR2⫹ monocyte/macrophages and tissue injury after kidney IRI was shown by protection from injury and reduced monocyte infiltration into injured tissue after adoptive transfer of CCR2⫺/⫺ monocytes to bone marrow–ablated mice. In contrast, reconstitution with CCR2⫹/⫹ monocytes facilitated
kidney macrophage infiltration and induced tissue injury. Therefore, inflammatory monocyte migration into the reperfused kidney is, at least in part, CCR2-dependent and contributes to the pathogenesis of early inflammation in kidney IRI. Fractalkine (CX3CL1) is a macrophage chemoattractant expressed on the surface of endothelial cells and is the ligand for the fractalkine receptor (CX3CR1). CX3CR1lowGR-1⫹CCR2⫹ subsets of monocytes actively are recruited to inflamed tissue whereas CX3CR1highGR-1⫺CCR2⫺ subsets of monocytes migrate to uninjured tissue
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and differentiate into resident macrophages and DCs.33 CX3CR1 deficiency (CX3CR1GFP/GFP mice; GFP, green fluorescent protein) or treatment with a CX3CR1-blocking antibody48 protected kidneys from IRI, thereby indicating that CX3CR1 is a key signal that mediates infiltration of inflammatory monocytes into injured kidneys after IRI. FUNCTIONAL ROLE OF MACROPHAGES AND DENDRITIC CELLS IN IRI Classic depletion and reconstitution studies have established the role of macrophages and DCs in renal injury.50,51 For example, nitrogen mustard or anti-macrophage serum was used to abrogate injury in a model of glomerulonephritis with the protective effect being lost after the reconstitution of macrophages.51 Macrophage depletion using bisphosphonate clodronate (dichloromethylene bisphosphonate) protected tissue from IRI,43,52 providing strong evidence for the participation of macrophages in the induction of injury after IRI. The role of DCs can be assessed more specifically through the use of transgenic mice expressing the human diphtheria toxin receptor (DTR; human heparin binding epidermal growth factor-like growth factor) in CD11c⫹ cells (CD11c-DTR transgenic mouse).53 Transgenic expression of the human DTR renders normally DT-resistant murine cells DT sensitive. By using this inducible lineage ablation method, exposure of CD11c-DTR mice to low-dose DT will kill primarily CD11c⫹ DCs. Kidney CD11c⫹GFP⫹ DCs were depleted efficiently in CD11c-DTR/GFP mice by DT but not by mutant DT, as revealed by immunofluorescence studies (Fig. 4A). Kidney injury, as indicated by an increase in plasma creatinine level (Fig. 4B) or H&E staining showing tubular cell necrosis (Fig. 4C), was significantly less in the CD11c-DTR/GFP mice that received DT treatment before IRI in comparison with control mutant DT treatment. The protective effect was not evident in DT- or mutant DT-treated wildtype control mice (Fig. 4B and C). Thus, DCs actively contribute to the early innate injurious response in kidney IRI.
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EFFECTOR MECHANISMS OF ANTIGEN-PRESENTING CELLS IN KIDNEY IRI
Activation of NKT Cells in Kidney IRI Although CD4⫹ cells are known to contribute to kidney IRI, the antigen-specific activation of T cells typically contributes to adaptive immunity and is a process not consistent with acute IRI that occurs within 24 hours. However, CD4⫹ T cells consist of functionally distinct subsets, including NKT cells that participate in innate immunity and could contribute to kidney IRI. CD4⫹ cells were found to infiltrate the kidney within 30 minutes after IRI (Fig. 2), and IFN-␥–producing CD1d-restricted NKT cells were identified early in kidney IRI. Furthermore, significant protection from kidney IRI was evident in mice administered a CD1d monoclonal antibody that blocks the interaction between antigen-presenting cells and NKT cells as well as mice deficient in NKT cells (Ja18⫺/⫺).54 These studies suggest that the CD1d molecule expressed on kidney DCs can present endogenous glycolipid to NKT cells (signal 1), thereby stimulating CD40-CD40L interaction (signal 2) and cytokine (IL-12) production (signal 3) to promote the innate immune response.55 Activation of CD1d-restricted NKT cells also promotes IFN-␥–producing GR-1⫹ neutrophil infiltration and tissue inflammation after kidney IRI.54
Soluble Mediators and Phagocytosis in Inflammation Macrophages and DCs are involved in antigen presentation and immunoregulation, and they express a large number of cell-surface proteins that play an important role in the phagocytic function of these cells. Activation of macrophages and DCs produces proinflammatory cytokines such as IL-1␣, IL-6, IL-12, IL-18, and TNF-␣ via MyD-88 – dependent or MyD-88 –independent pathways. The observation that kidneys produce IL-1 and IL-18 after IRI56 suggests the potential involvement of the inflammasome, a multiprotein complex in macrophages and DCs, in the inflammatory processes.57 NOD-like receptors possess pattern recognition receptors and are important sensors of microbial products and other pathogen-associ-
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Figure 4. DCs are important in the pathogenesis of kidney IRI. (A) CD11c⫹GFP⫹ cells (arrows) in mouse kidney sections from CD11c-DTR/GFP mice (which express the human DTR as a GFP fusion protein) were revealed immunohistochemically with GFP antibody (fluorescein isothiocyanate fluorescence, green). Biologically inactive mutant DT had no effect on the number of GFP-positive CD11c cells in the CD11c-DTR/GFP sham and IRI mouse kidneys. However, there were significantly less GFP-labeled cells in the sham and IRI kidneys of CD11c-DTR/GFP mice after administration of bioactive DT. Scale bar, 50 m. (B) Plasma creatinine level was measured in wild-type (WT) and CD11c-DTR/GFP mice that received mutant (control) or bioactive DT (4 g/g) 48 hours before surgery. Values are mean ⫾ SE; N ⫽ 2-11. ***P ⬍ .001. (C) H&E staining of kidneys from WT and CD11c-DTR/GFP mice reveals marked tubular injury in WT kidneys after IRI. Pretreatment with bioactive but not mutant DT before kidney IRI protected CD11c-DTR mouse kidneys from injury. Arrows indicate necrotic tubules. Scale bar, 100 m. (A and C) Data are representative of more than 3 experiments. Data from Li and Okusa, unpublished data, 2010.
ated molecular patterns. Once NOD-like receptors recognize microbial products or endogenous danger signals, this leads to activation of caspase 1 and assembly of the inflammasome. Caspase 1 then promotes the maturation of the inflammatory cytokines IL-1 and IL-18.58 No studies to date have examined the role of inflammasomes in acute kidney IRI. Insights into the mechanisms by which macrophages induce tissue injury may be gained from studies of their role in infection. The an-
timicrobial action of macrophages is mediated through oxidative mechanisms including the production of reactive oxygen intermediates by phagocyte oxidase and reactive nitrogen intermediates generated by inducible nitric oxide synthase 2(iNOS2).59,60 Macrophages also exert antimicrobial actions via a phagocyte oxidase– and nitric oxide synthase 2–independent mechanism.61 In addition, nonoxidative antimicrobial actions by macrophages may induce tissue injury. Macrophage-specific metalloelastase is in-
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volved in remodeling of the extracellular matrix associated with inflammation and the repair process.62 Macrophages also generate matrix metalloproteinase 12, a powerful enzyme that adheres to the bacterial wall to mediate bacterial killing but also degrades matrix proteins during inflammation. Thus, propensity of macrophages to elaborate these effective mediators for bacterial killing also may lead to tissue injury after the sterile inflammatory response that follows kidney IRI.
Macrophage and Dendritic Cell MicroRNAs in Inflammation MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by either degradation or translational repression of a target messenger RNA. They play a critical role in gene regulation through their stability and translation in the kidney.63,64 For example, uremia associated with acute kidney injury suppressed proinflammatory cytokine messenger RNA levels and altered the expression of at least 69 miRNAs.65 A number of miRNAs contribute to the maturation of macrophages via TLR signaling. TLR and IFN- stimulate production of miRNAs by the nuclear factor-B and Jun N-terminal kinase pathway. TNF-␣ is a target gene of miR-125b and down-regulation of miRNA-125b is required to ensure that a proper inflammatory response is generated by macrophages in response to microbial stimulation.66 The miRNA-146a reduces the expression of IRAK1 and TRAF6 of the TLR signaling cascade and thus acts to provide negative feedback to prevent excessive inflammation.67 Several TLR ligands increase miR-155 expression via the MyD88 or TRIF signal pathway and miR-155 is up-regulated by lipopolysaccharide and promotes TNF-␣ production from macrophages.68 Furthermore, miR-155 also regulates DC antigen presentation and costimulation activity with DCs from miR-155 knockout mice is unable to induce efficient T-cell activation.67 These studies show that a variety of miRNAs act to regulate DC and macrophage function and thus may participate in modulating tissue injury after kidney IRI.
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CONCLUSIONS DCs and macrophages are the most abundant leukocytes expressed in the normal kidney. They represent a continuum of cell phenotype in which macrophages, which have poor antigen processing and presentation properties, produce proinflammatory cytokines and soluble mediators that contribute to innate immunity in kidney IRI. At the other end of the spectrum are DCs, which, on activation, function in processing and presenting antigen to effector T cells. Presentation of endogenous glycolipid to NKT cells after kidney IRI initiates injurious responses. The early activation of DCs and macrophages orchestrates an immune response that leads to sterile inflammation and associated tissue damage and these immune cells likely will serve as important therapeutic targets in the future. ACKNOWLEDGMENTS The authors acknowledge Ms. Liping Huang and Ms. Hong Ye (Department of Medicine, University of Virginia) for generation of much of the data for this article; and Dr. Diane Rosin (Department of Pharmacology, University of Virginia) for helpful discussions and careful reading of the manuscript.
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