Multiple facets of macrophages in renal injury

Macrophage infiltration is a common feature of renal disease and their presence has been synonymous with tissue damage and progressive renal failure. More recently work has focused on the heterogeneity of macrophage activation and in particular their ability to curtail inflammation and restore normal function. This has led to the view that it is macrophage function rather than their number that is important in determining the outcome of inflammatory disease. This review will focus on the pathways that regulate macrophage infiltration and activation and how these could be manipulated to control renal inflammatory disease. In particular, the ability of specific cell surface receptors and intracellular signaling pathways to control macrophage activation and how macrophages can be genetically manipulated to develop properties that favor resolution over ongoing injury.     

Renal microenvironments and macrophage phenotypes determine progression or resolution of renal inflammation and fibrosis

Chronic kidney disease involves renal inflammation, interstitial fibrosis, and tubular and vascular atrophy. Macrophages seem to foster all of these histomorphological abnormalities, but their specific contributions remain controversial. Recruited monocytes differentiate into different tissue macrophage phenotypes, but current classifications are largely based on in vitro studies that do not adequately mirror tissue environments in vivo. To overcome this limitation, we propose to classify tissue macrophages according to their predominant roles in the phases of wound healing tissue environments, that is, inflammation, epithelial healing, mesenchymal healing, and fibrolysis. In this review, we discuss the evidence on respective macrophage phenotypes in renal pathology. This view sheds light on several aspects of renal remodeling in kidney disease: (1) renal infection or cell necrosis induces proinflammatory 'M1' macrophages that exacerbate renal cell damage, (2) uptake of apoptotic cells induces anti-inflammatory 'M2c/suppressor' macrophages that promote epithelial and vascular repair, (3) insufficient vascular and epithelial healing despite abundant growth factor secretion promotes profibrotic 'M2a/wound healing' macrophages that accelerate fibrogenesis, and (4) theoretically, fibrolytic macrophages should exist and await investigation.     

Dendritic cells and macrophages in kidney disease

Recent studies on dendritic cells (DCs) and macrophages have greatly advanced our knowledge of glomerular immunopathology. This rapidly developing field most likely has considerable impact on our understanding of the major mediators of tissue injury and repair in kidney disease. The pivotal role of cytokine in the initiation, differentiation, and amplification of local immune response production by antigen-presenting cells (APC; DCs, macrophages, and so forth) has been well documented, but the precise biological role of the numerous APC-derived products as effectors in both renal inflammation and repair remains a topic of intense research. This review focuses on the activated DCs and macrophages for the development and resolution of kidney disease, and discusses mechanistic information on the inflammatory process, including tissue injury and healing. A multi-dimensional study may contribute to further clarification of the role of their cellular activation in the progression of human kidney disease.     

Macrophages and progressive tubulointerstitial disease

Macrophages and progressive tubulointerstitial disease. In chronic renal disease, tubulointerstitial inflammation and injury is associated with infiltrating macrophages. As a consequence of primary injury, proteinuria, chronic hypoxia, and glomerular-derived cytokines may all differentially modulate the expression of factors that promote macrophage recruitment. In addition to adhesion molecules and chemokines, products of complement system and renin-angiotensin system activation may direct this process. Once present at interstitial sites, macrophages interact with resident cells and extracellular matrix to generate a proinflammatory microenvironment that amplifies tissues injury and promotes scarring. There is now increasing evidence for the efficacy of interventions directed against factors that recruit, activate, or are produced by macrophages. A detailed understanding of the biology of this area may lead to the further development of therapies that will improve the outcome of renal disease.     

Macrophages: A review of their role in wound healing and their therapeutic use

Macrophages are mononuclear phagocytes established during embryogenesis and derived from the yolk sac or the fetal liver but also recruited from the blood and bone marrow under proliferative inflammatory conditions (such as tissue repair). Most importantly, they take on distinct phenotypes and functions crucial to healing upon localization in the wound. The objective of this review is to summarize recent findings in regard to the cellular mechanisms of macrophages and chronic wounds. Advances in the potential use of macrophage therapy have arisen based, in part, on the fact that early recruitment of macrophages is critical to wound healing. Higher quality evidence is needed to support the use of macrophage therapy for chronic wound types, as is a better understanding of the signaling related to macrophage polarization, activation of macrophages, and their effect of mechanisms of repair. An evaluation of the currently available research on mechanism of action may lead to a better understanding of the signaling processes of the many macrophage phenotypes, as well as their roles and outcomes in wound healing, which could then guide the development and eventual widespread use of macrophage therapies.     

TLR2 and NODs1 and 2 cooperate in inflammatory responses associated with renal ischemia reperfusion injury

Pattern recognition receptors (PRRs) are potent triggers of tissue injury following renal ischemia/reperfusion injury (IRI). Specific PRRs, such as the toll-like receptor 2 (TLR2) and the nucleotide-binding oligomerization domain-like receptors (NLRs) NOD1 and NOD2 are promising targets to abrogate inflammatory injury associated with renal IRI. Several recent reports have shown there is crosstalk between TLRs and NODs, which might boost inflammatory responses to tissue injury. This study examined the relative roles of TLR2 and NODs 1 and 2 in activation of myeloid cells that contribute to inflammation after renal IRI. We found that TLR2 and NOD1 and 2 signaling induces neutrophil, macrophage and dendritic cell migration in vitro, however their blockade only decreases neutrophil infiltration into ischemic kidneys. The results of this study suggest that future therapies targeted to innate immune blockade should consider that either TLR2 or NOD1/2 blockade could decrease neutrophil inflammation following an ischemic insult to the kidney, however blockade of these PRRs would not likely impact infiltration of dendritic cells or macrophages. Developing rational approaches that target innate immunity in IRI-induced acute kidney injury requires an understanding of the relative role of PRRs in directing inflammation in the kidney.     

Macrophages contribute to the development of renal fibrosis following ischaemia/reperfusion-induced acute kidney injury.

BACKGROUND: Ischaemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function. Although most of the patients with acute kidney injury recover their renal function, significant portion of patients suffer from progressive deterioration of renal function. A persistent inflammatory response might be associated with long-term changes following acute ischaemia/reperfusion. Macrophages are known to infiltrate into tubulointersitium in animal models of chronic kidney disease. However, the role of macrophages in long-term changes after ischaemia/reperfusion remains unknown. We aimed to investigate the role of macrophages on the development of tubulointerstitial fibrosis and functional impairment following acute ischaemia/reperfusion injury by depleting macrophages with liposome clodronate. METHODS: Male Sprague-Dawley rats underwent right nephrectomy and clamping of left renal vascular pedicle or sham operation. Liposome clodronate or phosphate buffered saline was administered for 8 weeks. Biochemical and histological renal damage and gene expression of various cytokines were assessed at 4 and 8 weeks after ischaemia/reperfusion. RESULTS: Ischaemic/reperfusion injury resulted in persistent inflammation and tubulointerstital fibrosis with decreased creatinine clearance and increased urinary albumin excretion at 4 and 8 weeks. Macrophage depletion attenuated those changes. This beneficial effect was accompanied with a decrease in gene expression of inflammatory and profibrotic cytokines. CONCLUSIONS: These results suggest that macrophages play an important role in mediating persistent inflammation and fibrosis after ischaemia/reperfusion leading to a development of chronic kidney disease. Strategies targeting macrophage infiltration or activation can be useful in the prevention of development of chronic kidney disease following ischaemic injury.     

Macrophages play an essential role in trauma-induced sterile inflammation and tissue repair

Severe trauma is accompanied by a profound activation of the immune system. Patients with polytrauma develop systemic inflammatory response syndrome (SIRS) and often sepsis, which contributes substantially to high mortality of this condition. On a cellular level, necrosis and loss of plasma membrane integrity lead to the release of endogenous “damage-associated molecular patterns” (DAMPs) as danger signals, which in turn activate innate immune cells. Inflammation that occurs in the absence of invading pathogens has been termed sterile inflammation and trauma with tissue damage represents an acute form of sterile inflammation. Macrophages are a heterogeneous group of phagocytes of the innate immune system and serve as sentinels to detect loss of tissue integrity. Macrophages show a remarkable plasticity and undergo phenotypical changes in response to injury and repair. Under basal conditions, tissue-resident macrophages are distributed in various organ systems and have critical functions in tissue development and the maintenance of homeostasis. Inflammatory conditions, such as major trauma, lead to the rapid recruitment of blood-derived monocytes that mature into macrophages as well as direct recruitment of macrophages from the cavity that surrounds the injured organ. This leads to augmentation of the pool of tissue-resident macrophages. Besides their essential role in sensing tissue damage and initiating inflammation, macrophages contribution critically to tissue repair and wound healing, ultimately allowing full restoration. Dysregulated sterile inflammation and defective healing result in chronic inflammatory disease with persistent tissue damage. In this review, we summarize the cellular and molecular mechanisms that lead to activation of sterile inflammation, recruitment of immune cells and initiation of wound healing. We focus on the pivotal role of macrophages played in this context.     

The role of the macrophage in tendinopathy and tendon healing

The role of the macrophage is an area of emerging interest in tendinopathy and tendon healing. The macrophage has been found to play a key role in regulating the healing process of the healing tendon. The specific function of the macrophage depends on its functional phenotype. While the M1 macrophage phenotype exhibits a phagocytic and proinflammatory function, the M2 macrophage phenotype is associated with the resolution of inflammation and tissue deposition. Several studies have been conducted on animal models looking at enhancing or suppressing macrophage function, targeting specific phenotypes. These studies include the use of exogenous biological and pharmacological substances and more recently the use of transgenic and genetically modified animals. The outcomes of these studies have been promising. In particular, enhancement of M2 macrophage activity in the healing tendon of animal models have shown decreased scar formation, accelerated healing, decreased inflammation and even enhanced biomechanical strength. Currently our understanding of the role of the macrophage in tendinopathy and tendon healing is limited. Furthermore, the roles of therapies targeting macrophages to enhance tendon healing is unclear. Clinical Significance: An increased understanding of the significance of the macrophage and its functional phenotypes in the healing tendon may be the key to enhancing tendon healing. This review will present the current literature on the function of macrophages in tendinopathy and tendon healing and the potential of therapies targeting macrophages to enhance tendon healing.     

Macrophages in renal disease

Macrophages have heterogeneous phenotypes as they exercise their twofold role in the development and recovery of renal diseases. Some subpopulations of macrophages (M1) have a pathogenic function in renal inflammation, making them a logical target for elimination. Alternatively, M2 macrophage subpopulations resolve inflammation and repair injury, making them a potential therapeutic tool against renal injury. Here, we summarize recent findings regarding macrophage plasticity, and the various strategies for targeting or utilizing macrophages to treat renal disease. We highlight, in particular, the potential of renoprotective M2 macrophages to resolve inflammation and repair the kidney.     

GM-CSF Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury

After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4–stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury.     

Effect of estrogen and progesterone on macrophage activation during wound healing

Age-related impaired wound healing leads to substantial morbidity and mortality along with a large financial burden to health services. There is accumulating evidence that the tissue damage associated with chronic wounds is initiated and propagated by an inappropriately excessive inflammatory response. Research on age-related impaired wound healing suggests that the decline in sex steroid hormones with age may have a substantial influence on the inflammatory response in vivo. Topical and systemic estrogen treatments have shown an increased rate of healing by reducing inflammation, however the underlying mechanisms are little understood. In vitro studies also suggest progesterone may play a role in modulating inflammation. Macrophages are essential mediators of inflammation and wound healing. Macrophages can be activated in a classical or alternative manner in parallel with the T_H1/T_H2 dichotomy, respectively. Using a murine incisional wound healing model this study was carried out to investigate the roles of estrogen and progesterone on macrophage activation during the wound healing response. Our findings suggest with a reduction of steroid hormones following ovariectomy, alternatively activated macrophage markers (Fizz1 and Ym1) were reduced, with this effect being reversed with the administration of estrogen or progesterone; suggesting that with the reduction of steroid hormones macrophages are activated in a classical manner, promoting inflammation, whereas estrogen or progesterone are contributing toward macrophage activation in an alternative manner, driving wound repair, angiogenesis, and remodeling.     

Pro-resolution properties of macrophages in renal injury

Macrophages are inflammatory cells with important roles in the propagation of renal injury. More recently they have also been shown to be important in the resolution of inflammation. Wang et al. show that macrophages can be modulated ex vivo by cytokine stimulation, localize to renal tissue, and slow progression of experimental glomerular inflammation. Thus strategies targeting macrophage function have considerable therapeutic potential.     

Anti-inflammatory renoprotective effect of clopidogrel and irbesartan in chronic renal injury

Recent evidence suggests that platelet activation and angiotensin II may each contribute to glomerular inflammation and fibrosis. Clopidogrel inhibits platelet activation and may also reduce inflammation. This study investigated the anti-inflammatory and renoprotective effects of clopidogrel and irbesartan in the five-sixths nephrectomy rat model of chronic kidney disease. After 8 wk of treatment, 24-h proteinuria, serum creatinine, and histologic scores of glomerular sclerosis and tubulointerstitial damage were significantly lower in treated compared with untreated rats. Clopidogrel/irbesartan combination therapy had greater effects than either drug alone. Rats that underwent five-sixths nephrectomy had higher markers of platelet activation (plasma GMP-140 and renal cortical fibrin deposition) than sham-operated rats, and clopidogrel attenuated these effects. Clopidogrel and irbesartan similarly reduced the accumulation of ED-1-expressing macrophages in the cortical glomeruli and the interstitium. Combination therapy almost completely abolished macrophage infiltration and attenuated the expression of monocyte chemoattractant protein-1, intercellular adhesion molecule-1, TGF-beta(1), and connective tissue growth factor. In conclusion, combination treatment with clopidogrel and irbesartan, more so than either alone, decreases early renal injury induced by five-sixths nephrectomy by inhibiting renal inflammation.     

Interferon-gamma augments acute macrophage-mediated renal injury via a glucocorticoid-sensitive mechanism

Macrophages have been implicated in causing renal injury in both human and experimental kidney disease. The aim of the current study was to determine whether modulating the state of macrophage activation directly affects the capacity of these cells to cause renal injury. This was investigated using an adoptive transfer model in which macrophage activation can be manipulated in vitro, using interferon-gamma (IFN-gamma) or dexamethasone (Dex), and then macrophage-mediated renal injury determined in vivo. In this model, rats were made leukopenic by administration of cyclophosphamide (CyPh). Two days later (day 0), animals were injected with sheep anti-GBM serum followed by a single injection of rat NR8383 macrophages on day 1 and then killed 3 or 24 h after cell transfer. NR8383 macrophages were incubated IFN-gamma and/or Dex before adoptive transfer into animals. Induction of proteinuria and glomerular cell proliferation (PCNA+ cells) in this model was dependent on transfer of NR8383 macrophages. Exposure of macrophages to IFN-gamma for 18 h (but not 3 h) before transfer caused a twofold increase in the degree of proteinuria and glomerular cell proliferation compared with unstimulated cells (Nil versus IFN-gamma; P < 0.001). This was due to an increase in the number of transferred macrophages within the glomerulus and a significant increase in degree of renal injury per transferred glomerular macrophage. IFN-gamma increased iNOS and PDGF-B gene expression and upregulated adhesion molecule expression in NR8383 macrophages. In contrast, exposure of NR8383 cells to Dex for 18 h (but not 1 h) abrogated renal injury due to a failure of transferred macrophages to accumulate within the glomerulus. In addition, Dex abrogated renal injury caused by IFN-gamma-stimulated macrophages. In conclusion, activation of macrophages by IFN-gamma, independent of any effect on other leukocytes or renal cells, can substantially augment macrophage-mediated renal injury. This IFN-gamma augmentation of renal injury is sensitive to the action of glucocorticoids, which act directly on macrophages to prevent their recruitment to the inflamed glomerulus. This study provides the first evidence that it is possible to directly modulate macrophage-mediated renal injury.     

MCP-1 parallels inflammatory and regenerative responses in ischemic muscle

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) is important in macrophage recruitment and activation. However, the magnitude and temporal sequence of MCP-1 expression in relation to tissue injury and regeneration following ischemic injury remains unknown. MATERIALS AND METHODS: Hind limb ischemia was induced by femoral artery excision (FAE) in C57Bl/6J mice; a sham surgery was performed on the contralateral leg. Muscle lysates were used to measure MCP-1 and activities of creatine kinase, lactate dehydrogenase, and myeloperoxidase. Histology and immunohistochemistry were used to localize inflammation and MCP-1. RESULTS: FAE resulted in a prolonged period of ischemia and the administration of MCP-1 did not alter the restoration of perfusion. One day after femoral artery excision, extensive muscle necrosis and neutrophils were prevalent throughout the musculature of the lower leg. By 3 days, a mononuclear cell infiltrate predominated in association with robust muscle regeneration as indicated by myoD expression. Concomitantly, myeloperoxidase was maximally increased. Muscle enzymes (creatine kinase and lactate dehydrogenase) were maximally decreased within 3 days and returned to baseline levels by day 14, a time course consistent with injury and regeneration observed by histology. In parallel with these inflammatory and regenerative events, MCP-1 in muscle was maximally increased at day 3. By immunohistochemistry, MCP-1 was within vascular endothelial cells and infiltrating macrophages in areas of ischemic injury. CONCLUSIONS: The transient increases and selective tissue distribution of MCP-1 during early inflammation and muscle regeneration support the hypothesis that this cytokine participates in the early reparative events preceding the restoration of vascular perfusion following ischemic injury.     

Local macrophage proliferation in experimental Goodpasture's syndrome

Summary: The traditional dogma that macrophages do not proliferate within inflammatory lesions has recently been challenged. We have addressed this issue in a study of experimental Goodpasture's syndrome (rat anti-glomerular basement membrane [GBM] disease). Monocyte and macrophage proliferation was assessed during the initation and evolution of this severe inflammatory disease by expression of the proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation using twocolour immunohistochemistry. This study found that: (i) the initial accumulation of ED1⁺ macrophages in the kiney and lung seen during the indcution of disease resulted from blood monocyte recruitment; however, large numbers of proliferating macrophages (up to 60% of total macrophages) were present in these tissues during subsequent disease evolution; (ii) macrophage proliferation was restricted to the kidney and lung as demonstrated by the complete lack of PCNA expression and BrdU uptake by circulating monocytes and unchanged levels of resident macrophage proliferation within the spleen and liver; (iii) local macrophage proliferation within inflamed tissues was confined to cells of an ED1⁺ED2⁻ED3⁻ phenotype indicating that they were recently arrived monocytres and not resident tissue macrophages; and (iv) proliferating macrophages within inflamed tissues were localized in focal areas of severe tissue damage. In conclusion, this study has demonstrated that local proliferation of recuited monocytes makes a major contribution to macrophage accumulation within inflamed tissues during the evolution of rat anti-GBM disease. Furthermore, local macrophage proliferation may play an important role in the mediation of tissue injury in this disease model.     

Expression of the chemokines MCP-1/CCL2 and RANTES/CCL5 is differentially regulated by infiltrating inflammatory cells

BACKGROUND: Chemokines are involved in the regulation of the cellular renal infiltrate in glomerulonephritis; however, it is unclear to which degree resident glomerular cells or infiltrating leukocytes contribute to the formation of chemokines in glomerular inflammatory lesions. We therefore examined whether monocytes/macrophages play a role in the expression of the C-C chemokines MCP-1/CCL2 and RANTES/CCL5 in renal tissue in a lipopolysaccharide (LPS)-induced model of inflammation, where previously we have shown increased glomerular RANTES expression and glomerular infiltration of ED-1-positive cells. METHODS: Inflammatory lesions were induced by an intraperitoneal injection of LPS. The infiltration of monocytes into the glomerulus was reduced by two experimental approaches. First, rats were depleted of monocytes by the use of specific monocyte-antisera or by cytotoxic drugs. Second, the infiltration of monocytes into the kidney was reduced by using intercellular adhesion molecule-1 (ICAM-1) knockout mice. RESULTS: Both experimental approaches demonstrated a significant reduction in the number of infiltrating monocytes/macrophages after lipopolysaccharide injection. This reduction in the infiltration of inflammatory cells was associated with significantly reduced RANTES/CCL5 mRNA expression. However, MCP-1/CCL2 mRNA expression was not inhibited after the LPS injection by monocyte/macrophage depletion. Also, the increase in nuclear factor-kappaB (NF-kappaB) binding activity after the LPS injection was not reduced in pretreated animals. The experiments therefore demonstrate that infiltrating monocytes/macrophages contribute to increased RANTES/CCL5 mRNA expression in inflammatory renal lesions, whereas MCP-1/CCL2 mRNA expression and NF-kappaB activation were not reduced by monocyte/macrophage depletion. CONCLUSION: MCP-1/CCL2 released from renal tissue upon stimulation plays a major role in the regulation of monocyte/macrophage infiltration, which contributes significantly to increased renal RANTES/CCL5 expression. This cross-talk between resident renal cells and monocytes/macrophages is therefore likely to boost the number of infiltrating inflammatory cells.     

Granulocyte-macrophage colony-stimulating factor: Conductor of the wound healing orchestra?

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and is derived from both hemopoietic and nonhemopoietic sources which exert immunomodulatory properties. Various theories have been proposed to explain why some wounds become chronic and non-healing. Generalized suppression of inflammation locally or systemically may impede the body's physiological healing response by crippling the activity of reparative cells within the wound ecosystem. Thus, highlighting the importance of promoting host-directed therapeutics with immunomodulatory properties. The temporal and spatial expression of GM-CSF and GM-CSF receptors in the integumentary system suggests that epithelial-derived GM-CSF functions in an autocrine/paracrine manner. This may positively affect wound healing physiology via local inflammatory regulation promoting macrophage survival. Although diabetes negatively affects multiple aspects of wound healing GM-CSF activation is particularly impacted. Compared to acute/healthy wounds diabetic foot ulcers (DFU) only partially activate GM-CSF activity. There is a deleterious chain of events associated with this unfortunate sequala. DFUs also have a high proportion of monocytes and an absence of activated macrophages which results in an impaired inflammatory response. This may potentially serve as a vital point for GM-CSF to act as a companion diagnostic/theragnostic modality to help modulate the inflammatory response in wound healing. Correcting macrophage immune dysfunction with exogenous GM-CSF may help restore the immune balance in the wound ecosystem and jumpstart the wound healing cascade. Thus, the recognized beneficial role of GM-CSF in immune regulation across many studies provides a rationale for the initiation of the ongoing randomized controlled trials using GM-CSF.