Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease
2007; Elsevier BV; Volume: 72; Issue: 3 Linguagem: Inglês
10.1038/sj.ki.5002275
ISSN1523-1755
AutoresY. Wang, Y.P. Wang, Guoping Zheng, Vincent Lee, Linqi Ouyang, Dan Chang, Deepika Mahajan, Jana Coombs, Y.M. Wang, Stephen I. Alexander, David C.H. Harris,
Tópico(s)Reproductive System and Pregnancy
ResumoMacrophage infiltration of the kidney is a prominent feature associated with the severity of renal injury and progressive renal failure. To determine the influence of macrophages in renal disease models in the absence of endogenous T and B cells, we performed adoptive transfer of macrophages into severe combined immunodeficient (SCID) mice. In this study, macrophages were isolated from the spleens of BALB/c mice and stimulated with lipopolysaccharide to induce classically activated M1 macrophages or with interleukin-4 (IL-4) and IL-13 to induce alternatively activated M2 macrophages. These macrophages were then infused into SCID mice with adriamycin nephropathy; an in vivo model of chronic inflammatory renal disease analogous to human focal segmental glomerulosclerosis. Mice infused with M1 macrophages had a more severe histological and functional injury, whereas M2 macrophage-induced transfused mice had reduced histological and functional injury. Both M1 and M2 macrophages localized preferentially to the area of injury and maintained their phenotypes even after 4 weeks. The protective effect of M2 macrophages was associated with reduced accumulation and possibly downregulated chemokine and inflammatory cytokine expression of the host infiltrating macrophages. Our findings demonstrate that macrophages not only act as effectors of immune injury but can be induced to provide protection against immune injury. Macrophage infiltration of the kidney is a prominent feature associated with the severity of renal injury and progressive renal failure. To determine the influence of macrophages in renal disease models in the absence of endogenous T and B cells, we performed adoptive transfer of macrophages into severe combined immunodeficient (SCID) mice. In this study, macrophages were isolated from the spleens of BALB/c mice and stimulated with lipopolysaccharide to induce classically activated M1 macrophages or with interleukin-4 (IL-4) and IL-13 to induce alternatively activated M2 macrophages. These macrophages were then infused into SCID mice with adriamycin nephropathy; an in vivo model of chronic inflammatory renal disease analogous to human focal segmental glomerulosclerosis. Mice infused with M1 macrophages had a more severe histological and functional injury, whereas M2 macrophage-induced transfused mice had reduced histological and functional injury. Both M1 and M2 macrophages localized preferentially to the area of injury and maintained their phenotypes even after 4 weeks. The protective effect of M2 macrophages was associated with reduced accumulation and possibly downregulated chemokine and inflammatory cytokine expression of the host infiltrating macrophages. Our findings demonstrate that macrophages not only act as effectors of immune injury but can be induced to provide protection against immune injury. In human and animal renal disease, macrophage accumulation correlates with the degree of histological and functional injury, suggesting that macrophages play a role in renal injury.1.Eddy A.A. Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions.J Am Soc Nephrol. 1994; 5: 1273-1287Crossref PubMed Google Scholar, 2.Atkins R.C. Macrophages in renal injury.Am J Kidney Dis. 1998; 31: xlv-xlviiAbstract Full Text Full Text PDF PubMed Google Scholar, 3.Main I.W. Nikolic-Paterson D.J. Atkins R.C. T cells and macrophages and their role in renal injury.Semin Nephrol. 1992; 12: 395-407PubMed Google Scholar Adriamycin (ADR)-–induced nephropathy (AN) is a robust experimental analog of human focal segmental glomerulosclerosis, characterized by changes in both tubulointerstitial and glomerular compartments. This animal model of AN was initially established in rat. We previously developed a model of AN in BALB/c mouse, which was similar to AN in rat. More recently, a model of AN was developed in severe combined immunodeficiency (SCID) mice which are homozygous for an autosomal recessive mutation leading to the absence of lymphocytes. In spite of the absence of lymphocytes, SCID mice developed functional and histological changes similar to those of immunocompetent mice, yet with increased sensitivity to ADR-induced injury.4.Lee V.W. Wang Y. Qin X. et al.Adriamycin nephropathy in severe combined immunodeficient (SCID) mice.Nephrol Dial Transplant. 2006; 21: 3293-3298Crossref PubMed Scopus (25) Google Scholar Antibody depletion of macrophages (using the monoclonal antibody ED7) resulted in reduced renal injury in AN.5.Wang Y. Mahajan D. Tay Y.C. et al.Partial depletion of macrophages by ED7 reduces renal injury in Adriamycin nephropathy.Nephrology (Carlton). 2005; 10: 470-477Crossref PubMed Scopus (28) Google Scholar Ablation of macrophages in murine crescentic glomerulonephritis also reduced renal injury and improved renal function.6.Duffield J.S. Tipping P.G. Kipari T. et al.Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 167: 1207-1219Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar Our recent studies in SCID mice (deficient in T and B cells) and NOD/SCID mice (deficient in T, B, and NK cells) have shown that neither lymphocytes nor NK cells are necessary for induction of this model, consistent with a primary role for macrophages as key effectors in AN.7.Zheng G. Zheng L. Wang Y. et al.NK cells do not mediate renal injury in murine adriamycin nephropathy.Kidney Int. 2006; 69: 1159-1165Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Other studies have instead shown a beneficial role for macrophages, which depends on their phenotype and location and the nature of injury.8.Erwig L.P. Kluth D.C. Rees A.J. Macrophage heterogeneity in renal inflammation.Nephrol Dial Transplant. 2003; 18: 1962-1965Crossref PubMed Scopus (14) Google Scholar, 9.Friedman S.L. Mac the knife? Macrophages – the double-edged sword of hepatic fibrosis.J Clin Invest. 2005; 115: 29-32Crossref PubMed Scopus (80) Google Scholar, 10.Sutterwala F.S. Noel G.J. Salgame P. et al.Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I.J Exp Med. 1998; 188: 217-222Crossref PubMed Scopus (263) Google Scholar, 11.Herbert D.R. Holscher C. Mohrs M. et al.Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology.Immunity. 2004; 20: 623-635Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar In mice with unilateral ureteric obstruction (a model of chronic renal injury) reconstituted with bone marrow of angiotensin II type 1 receptor gene knockout mice, infiltrating macrophages were shown to play an antifibrotic role.12.Nishida M. Fujinaka H. Matsusaka T. et al.Absence of angiotensin II type 1 receptor in bone marrow-derived cells is detrimental in the evolution of renal fibrosis.J Clin Invest. 2002; 110: 1859-1868Crossref PubMed Scopus (113) Google Scholar Macrophages transfected with adenovirus to express IL-4 have been shown to reduce renal inflammation in rats with nephrotoxic nephritis.13.Kluth D.C. Ainslie C.V. Pearce W.P. et al.Macrophages transfected with adenovirus to express IL-4 reduce inflammation in experimental glomerulonephritis.J Immunol. 2001; 166: 4728-4736Crossref PubMed Scopus (74) Google Scholar The capacity to modify macrophage function has been demonstrated in a number of studies in vitro.14.Ma J. Chen T. Mandelin J. et al.Regulation of macrophage activation.Cell Mol Life Sci. 2003; 60: 2334-2346Crossref PubMed Scopus (267) Google Scholar, 15.Katakura T. Miyazaki M. Kobayashi M. et al.CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages.J Immunol. 2004; 172: 1407-1413Crossref PubMed Scopus (130) Google Scholar, 16.Zhang J. Tachado S.D. Patel N. et al.Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.J Leukoc Biol. 2005; 78: 665-674Crossref PubMed Scopus (78) Google Scholar, 17.Stumpo R. Kauer M. 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Alternative activation of macrophages.Nat Rev Immunol. 2003; 3: 23-35Crossref PubMed Scopus (4379) Google Scholar Lipopolysaccharide and proinflammatory stimuli such as interferon-γ or tumor necrosis factor-α (TNF-α) induce classically activated macrophages (M1 macrophages), characterized by antimicrobial and cytotoxic properties, which underlie their role in host responses to infection or autoimmune disease, whereas exposure to IL-4 and IL-13 results in alternative activation of macrophages (M2 macrophages). Recent studies have shown that these alternatively activated cells produce several components involved in the synthesis of extracellular matrix, suggesting that their primary role may involve tissue repair rather than microbial killing.22.Gratchev A. Guillot P. Hakiy N. et al.Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3.Scand J Immunol. 2001; 53: 386-392Crossref PubMed Scopus (217) Google Scholar M2 macrophages have been studied extensively in vitro with respect to their suppressive activity, secretion of anti-inflammatory cytokines and ability to modulate wound healing and angiogenesis.23.Mosser D.M. The many faces of macrophage activation.J Leukoc Biol. 2003; 73: 209-212Crossref PubMed Scopus (1359) Google Scholar However, adoptive transfer of cytokine-programmed M2 macrophages has not been explored in vivo. We hypothesized that macrophages could be polarized in vitro in a way that would result in a persistent in vivo functional phenotype. Using SCID mice, we evaluated the effect of different macrophage phenotypes on tissue injury in vivo in the absence of T and B cells. Transfusion of M1 macrophages not surprisingly exacerbated AN, whereas M2 macrophages were shown to protect against in vivo renal injury in the presence of host macrophages suggesting they could exert a dominant protective effect. Macrophages were isolated from the spleen of BALB/c mice using a magnetic bead isolation kit with CD11b beads. The fraction of CD11b-positive cells from fresh spleens following purification was 96%, whereas CD11c (dendritic cell marker), CD19 (B-cell marker), and CD49b (NK-cell marker)-positive cells accounted for <1% of the purified suspension (Figure 1). There was strong expression of CCL3, inducible nitric oxide synthase (iNOS), TNF-α, CD86, and major histology complex class II by M1 macrophages, but not by M0 (nonstimulated macrophages) or M2 macrophages. In contrast, expression of CCL17, mannose receptor, and secretion of IL-10 were seen with M2 macrophages, but not M1 macrophages. Trace expression of mannose receptor was seen with M0 macrophages (Figure 2). We sought to investigate the effect of adoptive transfer of ex vivo-modulated macrophages in established renal injury. AN was induced in SCID mice by tail-vein injection of 5.2 mg/kg ADR. Macrophages (M0, M1, or M2 – see Materials and Methods) were infused by tail-vein injection 5 days after ADR. Serum creatinine and urine protein were assessed at days 14, 21, and 28 after ADR, and were significantly worse following transfusion with M1 macrophages, significantly better with M2 macrophages, and unchanged with M0 macrophages compared with AN alone at day 28 (Figure 3). Transfusion of culture medium from final stages of M0, M1, or M2 macrophage preparation (before their transfusion) had no significant effect on kidney function or structure in normal or AN mice (data not shown). Marked renal injury was seen at week 4 in AN, characterized by glomerular sclerosis, tubular atrophy, and interstitial fibrosis with significant mononuclear cell infiltration. Renal injury was increased greatly in AN mice transfused with M1 macrophages compared with control AN mice. Conversely, injury was significantly reduced in mice transfused with M2 macrophages, whereas there were no significant changes in mice transfused with M0 macrophages. The number of points within trichrome-positive vs trichrome-negative cortical fields were counted at × 200 magnification from randomly selected 10 fields, and expressed as a percentage. In parallel with the tubulointerstitial injury findings, M1 macrophage transfusion induced more fibrosis (45.4±4.5%; P<0.01) as compared with AN control (29.2±5.1%) and AN transfused with M0 macrophages (27.7±3.9%), and M2 macrophage transfusion significantly suppressed the development of interstitial fibrosis (15.1±3.1%; P<0.01 vs other three groups) (Figure 4a–e). Tissue sections were double stained with immunofluorescent antibodies F4/80 for macrophages and Ki67 for proliferative cells. Very few double-positive cells were observed. Single immunohistochemical staining with F4/80 and Ki67 showed an increase in the number of total F4/80-positive cells and Ki67-positive nuclei in the M1 transfused mice and a significant reduction in the M2 transfused mice compared with the M0 transfused mice (Figure 5). The quantitation of these positive cell numbers is shown in Table 1.Table 1Quantitative analysis of F4/80-positive and Ki67-positive cellsAN+vehicleAN+M0AN+M1AN+M2F4/80-positive cells84.17±7.7386.67±8.89126.71±17.46aP<0.01 compared with other three groups.50.83±6.35bP<0.05 compared with other three groups.Ki67-positive cells15.37±2.1816.98±2.7322.68±3.54bP<0.05 compared with other three groups.9.23±1.56bP<0.05 compared with other three groups.The values represent the mean±s.d. of positive cells/ × 400 field.a P<0.01 compared with other three groups.b P<0.05 compared with other three groups. Open table in a new tab The values represent the mean±s.d. of positive cells/ × 400 field. One million fluorescently labeled M0, M1, or M2 macrophages were injected via tail vein at day 5 after ADR or into control normal mice. Fluorescently labeled cells were distributed to kidney, spleen, and liver of AN mice 24 h after transfer (day 6). Interestingly, there was no trace of fluorescently labeled cells in lung and heart 24 h after transfusion. Few of these cells were seen in spleen and liver at days 14 and 28 (Figure 6a), whereas transfused macrophages accumulated progressively in kidneys up to day 28 after ADR. A reduction of native macrophage infiltration was observed in mice transfused with M2 macrophages. (Figure 6b–f). The above in vivo experiment demonstrated renal accumulation of transfused macrophages and especially M1 macrophages as long as 4 weeks after transfusion. The number and viability of macrophages were examined. The cell numbers decreased to around half those of the initial seeding (from 1.3 × 107 to 6.5 × 106) after 28 days culture in vitro. The viability of the cells as defined by trypan blue staining was approximately 85% after 28 days of in vitro culture. Similarly, the proportion of apoptotic cells after 28 days increased to 18–25% by annexin V staining. Therefore, we next tested the stability of macrophage phenotype over time. We compared the expression of CCL3 and iNOS mRNA by freshly separated M1 to that of M1 macrophages cultured for 4 weeks, using real-time polymerase chain reaction (PCR). In vitro expression of CCL3 and iNOS by M1 macrophages was maintained for 4 weeks. Similarly, the expression of CCL17 and mannose receptor mRNA by M2 macrophages was maintained for 4 weeks (Figure 7). Macrophages were isolated from AN kidneys at day 28, using saline perfusion and CD11b beads. Macrophage iNOS, CCL3, CCL17, mannose receptor, CCL2, and TNF-α mRNA expression were assessed. There was a trend toward decreased total macrophage mRNA expression of CCL2 and TNF-α after M2 macrophage transfusion. In contrast, renal macrophage mRNA expression of iNOS, CCL2, and TNF-α expression was significantly upregulated after M1 macrophage transfusion. Renal macrophage mRNA expression of CCL17 at day 28 was upregulated after M2 macrophage transfusion (Figure 8). No difference was seen in expression of mannose receptor and CCL3 among all groups (data not shown). In this study we examined the effect of transfused macrophages programmed ex vivo by cytokines, in a model of chronic inflammatory renal disease (AN). Transfusion of M0 macrophages had no significant effect on renal injury. M1 macrophages promoted both histological damage and functional impairment. However, M2 macrophages strongly protected renal structure and function. Once modified to a M1 or M2 phenotype, macrophages maintained their properties in vitro for up to 4 weeks. This provides proof of the potential of macrophages cultured with IL-4 and IL-13 as a treatment for chronic renal disease. The harmful effect of preactivated macrophages has been demonstrated previously, in models of kidney disease. Ikezumi et al.24.Ikezumi Y. Atkins R.C. Nikolic-Paterson D.J. Interferon-gamma augments acute macrophage-mediated renal injury via a glucocorticoid-sensitive mechanism.J Am Soc Nephrol. 2003; 14: 888-898Crossref PubMed Scopus (61) Google Scholar demonstrated that adoptive transfer of macrophages incubated with interferon-γ caused a twofold increase in the degree of proteinuria and glomerular cell proliferation compared with transfer of unstimulated macrophages in a cognate model of renal disease, antiglomerular basement membrane nephritis. This finding is in line with our results that demonstrated an augmentation of renal damage by M1 macrophages in AN in SCID mice, a model of innate immune renal injury. Ikezumi et al.,25.Ikezumi Y. Hurst L.A. Masaki T. et al.Adoptive transfer studies demonstrate that macrophages can induce proteinuria and mesangial cell proliferation.Kidney Int. 2003; 63: 83-95Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar in a separate study, observed induction of proteinuria and mesangial cell proliferation by adoptive transfer of M0 macrophages during the acute stage of rat accelerated antiglomerular basement membrane disease. The absence of such an effect of M0 macrophages in AN may relate to the primary cellular nature of the inflammation in AN vs the antibody-driven pathology of antiglomerular basement membrane disease where even M0 macrophages may be activated through Fc receptors by pathogenic antibodies. The most striking finding in these studies is that M2 macrophages protected against both structural and functional damage in AN, a protective effect which has only previously been demonstrated in vitro.10.Sutterwala F.S. Noel G.J. 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La Flamme A.C. et al.Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.J Immunol. 2001; 167: 6533-6544Crossref PubMed Scopus (537) Google Scholar Other studies have suggested that microenvironment can reprogram the functional phenotype of macrophages.30.Stout R.D. Jiang C. Matta B. et al.Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences.J Immunol. 2005; 175: 342-349Crossref PubMed Scopus (639) Google Scholar Our results provide in vivo support for the suggestion that macrophage phenotype persists despite the microenvironment. We found that both M1 and M2 macrophages trafficked to inflamed kidneys in AN. This suggests that the expression of chemokine and other trafficking receptors by these macrophages has the potential to deliver them to sites of inflammation. We have previously shown in several models of renal disease the expression of a variety of chemokines including the CCL2, a monocyte recruiting chemokine. This targeting to the inflamed organ enhances efficacy relative to the number of cells transferred and provides promise for potential clinical studies. Other strategies to ameliorate impaired renal function in animal models have included direct cytokine gene transfer, chemokine blockade, or macrophage manipulation.31.Wilson H.M. Kluth D.C. Targeting genetically modified macrophages to the glomerulus.Nephron Exp Nephrol. 2003; 94: e113-e118Crossref PubMed Scopus (4) Google Scholar, 32.Yang J. Reutzel-Selke A. 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Jobin C. et al.Inhibition of macrophage nuclear factor-kappaB leads to a dominant anti-inflammatory phenotype that attenuates glomerular inflammation in vivo.Am J Pathol. 2005; 167: 27-37Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Cytokine manipulation of macrophages to the M2 phenotype provides many of the advantages of gene transduction without the associated risks. The advantages of this method include the simplicity of in vitro macrophage modulation by cytokines, and the effectiveness of intravenous administration without the need for specialized delivery. Most important is the fact that it avoids the limitations of gene therapy including gene delivery and risks of the viral vector.40.Wells C.A. Ravasi T. Faulkner G.J. et al.Genetic control of the innate immune response.BMC Immunol. 2003; 4: 5Crossref PubMed Scopus (110) Google Scholar,41.Polak J. Hench L. Gene therapy progress and prospects: in tissue engineering.Gene Ther. 2005; 24: 1725-1733Crossref Scopus (42) Google Scholar Previous studies of renal macrophage phenotype have studied the composition of the macrophage population. In the anti-Thy1.1 model of proliferative nephritis, 70% of macrophages infiltrating glomeruli demonstrated a M1 phenotype and 30% behaved as if activated by transforming growth factor-β.42.Minto A.W. Erwig L.P. Rees A.J. Heterogeneity of macrophage activation in anti-Thy-1.1 nephritis.Am J Pathol. 2003; 163: 2033-2041Abstract Full Text Full Text PDF PubMed Google Scholar Our study was able to do this in a very informative way. Having demonstrated that short-term induction of M1 or M2 phenotype led to a persistence of these characteristics for up to 4 weeks in culture, we evaluated the effect of transfer of these cells on host macrophages. M1 transfused mice exhibited skewing of expression of host macrophage-related molecules to that of the transfused population. However, M2 macrophage transfusion caused an apparent (nonsignificant) decrease in expression of CCL2 and TNF-α and a significant increase in expression of CCL17 in total macrophages suggesting that in addition to their possible effects on other cells M2 macrophages could skew the host macrophage phenotype. M1 macrophages are known to have direct effects on renal parenchymal cells via the release of NO and other mediators,43.Duffield J.S. Erwig L.P. Wei X. et al.Activated macrophages direct apoptosis and suppress mitosis of mesangial cells.J Immunol. 2000; 164: 2110-2119Crossref PubMed Scopus (103) Google Scholar whereas it is unknown whether M2 macrophages can directly affect the parenchyma. Thus, it is possible that the protective effect of M2 macrophages may involve a direct or indirect effect on renal parenchymal cells, in particular tubular epithelial and mesangial cells. In conclusion, this study provides the direct evidence that macrophage modulation ex vivo by cytokines to an anti-inflammatory phenotype (M2), is an effective strategy for treating experimental chronic inflammatory renal disease. The regulatory effects of M2 macrophage were shown to be target-specific, sustainable, and dominant. Six- to eight-week-old male SCID and BALB/c mice obtained from the Animal Resources Centre (Perth, Australia) were used in this study. The Animal Ethics Committee of Westmead Hospital approved all procedures. Dose-finding studies defined an optimal dose of 5.2 mg/kg body weight of ADR (Pharmacia & Upjohn Pty Ltd., Australia). ADR was injected once via the tail vein of each non-anesthetized SCID mouse.44.Wang Y. Wang Y.P. Tay Y.C. et al.Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.Kidney Int. 2000; 58: 1797-1804Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar BALB/c mice splenocytes were harvested and washed in ice-cold RPMI 1640 medium (Invitrogen, Mount Waverly, Australia). Tissue was triturated with the sterile syringes, and the resulting cell suspension was filtered through 40-μm nylon mesh (BD Biosciences, North Ryde, Australia) and then incubated at 37°C for 30 min. The adherent cells were harvested and purified by MACS CD11b+ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, Bergisch Gladbach). These spleen-derived macrophages were rinsed three times with RPMI 1640 medium and further processed to become M0, M1, and M2. Macrophages cultured in the normal medium for 48 h were defined as M0; macrophages undergoing a 2-h incubation with lipopolysaccharide (2.5 μg/ml) and then cultured in normal medium for another 46 h were defined as M1; and macrophages undergoing the incubation with IL-4/13 (10 ng/ml each) for 48 h were defined as M2. Lipopolysaccharide was purchased from Sigma-Aldrich (Castle Hill, Australia), and IL-4 and IL-13 were purchased from Invitrogen. Apoptosis was analyzed by flow cytometric detection of fluorescein isothiocyanate (FITC)-labeled annexin V (BD, North Ryde, Australia) according to the company’s manual. In brief, collected macrophages were washed twice with cold phosphate-buffered saline and then cells were resuspended in 1 × binding buffer at a concentration of 1 × 106 cells/ml. Then 100 μl of the solution (1 × 105 cells) was incubated for 15 min at room temperature (25°C) in the dark after adding 5 μl of annexin V-FITC and 5 μl of propidium iodide. After that, 400 μl of 1 × binding buffer was added to each tube. Flow cytometric analy
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