The origin, function, and sometimes even the existence of macrophages in the brain have been vigorously debated over the past century (see, for example, the historical overview by Peters, Palay, and Webster in The Fine Structure of the Nervous System, Oxford, 1991). Many issues have been resolved in recent years, but the cells remain surprisingly refractory to scientific interrogation. In Alzheimer disease, reactive microglia are a prominent cellular component of senile plaques, and hence, they have attracted the attention of researchers who wish to establish whether they are harmful or beneficial. The microglia themselves furnish evidence to support both views: As macrophages, they are equipped to rid the brain of unwanted material, yet this capability also gives them the potential to do collateral damage in the process.

This intriguing paper by Simard, Rivest, and colleagues provides evidence for a beneficial role of microglia in removing excess β-amyloid in vivo. Their data indicate, in plaque-producing transgenic mice (including a model that also expresses thymidine...  Read more

The origin, function, and sometimes even the existence of macrophages in the brain have been vigorously debated over the past century (see, for example, the historical overview by Peters, Palay, and Webster in The Fine Structure of the Nervous System, Oxford, 1991). Many issues have been resolved in recent years, but the cells remain surprisingly refractory to scientific interrogation. In Alzheimer disease, reactive microglia are a prominent cellular component of senile plaques, and hence, they have attracted the attention of researchers who wish to establish whether they are harmful or beneficial. The microglia themselves furnish evidence to support both views: As macrophages, they are equipped to rid the brain of unwanted material, yet this capability also gives them the potential to do collateral damage in the process.

This intriguing paper by Simard, Rivest, and colleagues provides evidence for a beneficial role of microglia in removing excess β-amyloid in vivo. Their data indicate, in plaque-producing transgenic mice (including a model that also expresses thymidine kinase), that many macrophages associated with relatively mature senile plaques originate from the periphery, and that it is these cells, rather than the resident microglia, that phagocytose Aβ and thereby lower plaque load. The bone marrow-derived macrophages are summoned to dispose only of senile plaques that have evolved to the point where, presumably, they present a distinct threat to the integrity of the brain. This finding supports the view that activated microglia probably do not mediate the early deposition of Aβ in APP-transgenic mice (Stalder et al., 1999) or in aged humans (Vogelgesang et al., 2002). It also suggests a tentative resolution to the question of what microglia are doing in plaques: It depends in part on the origin of the cells. On balance, these interesting data in mice imply that promoting the phagocytosis of brain amyloid by bone marrow-derived microglia could reduce plaque load in AD.

That said, provoking a cellular attack on Aβ is a potentially high-reward but also risky strategy, as has been made clear by the Aβ immunization trials for AD. With this in mind, there are some additional issues that might be considered before calling on peripheral macrophages to join the fray. First, the relevance of Aβ clearance in transgenic mice to the situation in human AD remains uncertain, as there is considerable evidence for critical differences between murine and human senile plaques/Aβ deposits, and possibly in the way that the two species respond immunologically to Aβ. Second, it is important to repeat this study in larger numbers of mice, and preferably in male and female mice, which can differ significantly in their tendency to deposit Aβ. Studies in biologically intermediate species might help to bridge this gap. In addition, it would be beneficial to know if, for example, oligomeric Aβ, tau, and neuronal integrity are affected by blood-borne microglia in animal models. Ultimately, behavioral improvement will be the arbiter of the value of any potential therapy.

It would be ideal to impede the Aβ-cascade before the toxic effects of Aβ become manifest in the brain. If, however, recruitment of peripheral macrophages can be demonstrated to be selective, safe, and effective in humans, this approach could indicate a path to treating AD at a point in the pathogenesis of the disorder when other types of therapy might be too late.

View all comments by Lary Walker

This is an interesting manuscript. It confirms an earlier study by Wisniewski et al., 1991, which reported that invading macrophages after brain injury phagocytose amyloid while resident microglia appear not to do so. However, the question of whether a CNS lesion, that is, disruption of the blood-brain barrier (BBB), is necessary for a “phagocytotic” activity of invading macrophages still remains unanswered in this new study. That is because Simard and collaborators inserted a catheter into the ventricle, which obviously affected the integrity of the BBB.

The authors also suggest amyloid phagocytosis of the invading macrophages based on co-staining of a lysosomal marker with Aβ. Unfortunately this co-staining was not done for resident microglia. Colocalization of Aβ/amyloid at the level of confocal microscopy does not unequivocally prove amyloid phagocystosis (see, e.g., Wisniewski et al. above; Stalder et al., 2001). Because the role of resident microglia was not studied, further...  Read more

This is an interesting manuscript. It confirms an earlier study by Wisniewski et al., 1991, which reported that invading macrophages after brain injury phagocytose amyloid while resident microglia appear not to do so. However, the question of whether a CNS lesion, that is, disruption of the blood-brain barrier (BBB), is necessary for a “phagocytotic” activity of invading macrophages still remains unanswered in this new study. That is because Simard and collaborators inserted a catheter into the ventricle, which obviously affected the integrity of the BBB.

The authors also suggest amyloid phagocytosis of the invading macrophages based on co-staining of a lysosomal marker with Aβ. Unfortunately this co-staining was not done for resident microglia. Colocalization of Aβ/amyloid at the level of confocal microscopy does not unequivocally prove amyloid phagocystosis (see, e.g., Wisniewski et al. above; Stalder et al., 2001). Because the role of resident microglia was not studied, further work is needed to elucidate the function and impact of resident microglia versus invading macrophages.

View all comments by Mathias Jucker

I would like to thank Erene Mina and Drs. Walker and Jucker. They provide insightful comments regarding specific aspects of the study. I'd like to address a few points here.

The first one regards irradiation and its effects on the blood-brain barrier (BBB). There is not very strong evidence that irradiation alters the BBB, and brain infiltration of bone marrow-derived cells has been reported with other techniques as well. Messengale and colleagues have validated this concept using both lethal irradiation and parabiosis techniques in mice (Massengale et al., 2005). Although most (if not all) GFP cells found in the brains of chimeric mice have a microglial phenotype, the overall contributions of such cells to the brain-resident microglial populations of normal mice remain quite low (e.g., 0.5-11.5 percent of resident microglia). This is what we generally observe in our mice (Simard and Rivest, 2004). In APP mice, however, there is a robust microglial recruitment toward the plaques, and those that derive from the bone marrow are attracted at a specific time of the disease....  Read more

I would like to thank Erene Mina and Drs. Walker and Jucker. They provide insightful comments regarding specific aspects of the study. I'd like to address a few points here.

The first one regards irradiation and its effects on the blood-brain barrier (BBB). There is not very strong evidence that irradiation alters the BBB, and brain infiltration of bone marrow-derived cells has been reported with other techniques as well. Messengale and colleagues have validated this concept using both lethal irradiation and parabiosis techniques in mice (Massengale et al., 2005). Although most (if not all) GFP cells found in the brains of chimeric mice have a microglial phenotype, the overall contributions of such cells to the brain-resident microglial populations of normal mice remain quite low (e.g., 0.5-11.5 percent of resident microglia). This is what we generally observe in our mice (Simard and Rivest, 2004). In APP mice, however, there is a robust microglial recruitment toward the plaques, and those that derive from the bone marrow are attracted at a specific time of the disease. Other groups have observed a similar pattern in irradiated APP mice (Malm et al., 2005; Stalder et al., 2005), and one can appreciate the robust microglia infiltration in the plaques of non-irradiated mice (Fig. 1 and supp. movie 1). Therefore, I do not think that infiltration is caused by alteration of the BBB in irradiated mice, but is a natural process that is especially dynamic while the plaques progress. The mechanisms explaining why bone marrow-derived microglia are no longer recruited toward the plaques at a specific time point in the disease have yet to be unraveled with future experiments.

Another point raised is that we did not look at the colocalization of Aβ in the lysosomes of the resident microglia. We actually did a meticulous analysis of such processes in the chimeric APP, and while GFP cells were almost always associated with lysosomal Aβ, the resident cells were not. This is the reason that we did not show these results, but we have discussed them.

We observed phagocytosis by bone marrow-derived microglia during a very specific time. This takes place around 6 months of age in the APP/PS1 mice, and we no longer see these cells at 9 months. Therefore, cell recruitment (of bone marrow origin) and phagocytosis are dynamic and transient phenomena, which may explain why other groups have not detected it. This also explains why inhibition of cell recruitment (APP/TK mice) from 5 to 6 months has such profound consequences on plaque growth. We are now working on new genetic strategies to enhance and improve the recruitment of these cells for a longer period of time to see if we can prevent the amyloid cascade and cognitive deficit.

Finally, multiple staining and 3D reconstructions using confocal laser-scanning microscopy are powerful tools to determine cellular compartmentalization, such as Aβ within the lysosomal GFP cells.

References:
Malm TM, Koistinaho M, Parepalo M, Vatanen T, Ooka A, Karlsson S, Koistinaho J. Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to β-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol Dis. 2005 Feb;18(1):134-42. Abstract

Massengale M, Wagers AJ, Vogel H, Weissman IL. Hematopoietic cells maintain hematopoietic fates upon entering the brain. J Exp Med. 2005 May 16;201(10):1579-89. Abstract

Simard AR, Rivest S. Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J. 2004 Jun;18(9):998-1000. Epub 2004 Apr 14. Abstract

Simard AR, Soulet D, Gowing G, Julien JP, Rivest S. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 2006 Feb 16;49(4):489-502. Abstract

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

View all comments by Serge Rivest

I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while...  Read more

I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while opposing the proinflammatory response, both of which likely exist as a continuum of innate immune cell activation profiles (Town et al., 2005). But, if we can accomplish this, will amyloid-reducing therapies ultimately be successful AD therapeutics? As stated by Dave Morgan and others on this forum, the first test of the amyloid cascade hypothesis of AD in humans will likely be the Aβ vaccine. We anxiously await whether the hypothesis holds up and delivers an efficacious AD therapy. If it does, then the floodgates will open for a whole host of amyloid-targeted AD therapeutics—both immune and non-immune.

About the issue raised by Mathias Jucker and Tony Wyss-Coray of CD11c as a marker for blood-borne innate immune cells/macrophages versus microglia, I should mention that we initially thought that CD11c would be a microglial marker in the context of AD. However, after examining numerous brain sections from various ages of wild-type versus Tg2576 or mutant APP/PS1 doubly transgenic mice for CD11c expression, we concluded that while microglia in the parenchyma around Aβ deposits were CD11b, CD45, MHC II, F4/80 Ag, and CD68 positive, they were negative for CD11c. However, we did observe a small number of round, non-process bearing CD11c positive cells within the lumen of blood vessels in both Tg2576 and APP/PS1 mice, consistent with Stalder and colleagues’ report of invading hematopoietic cells in brains of aged Tg2576 mice. At the time that we were checking for CD11c expression in AD mice, Alon Monsonego and Harold Weiner published a review in Science where they mentioned (as data not shown) that plaque-associated microglia were CD11c positive. I called Alon and asked him about the methodological details. However, after trying various tissue handling techniques, antibodies, and confocal settings, I was unable to reproduce this despite getting microglia in day 20 MOG-EAE brain sections to light up like a Christmas tree with CD11c. I came away thinking that it is possible to acutely activate microglia with the necessary vigor to promote CD11c expression, for example, in the context of EAE. However, I believe that this form of activation does not occur in AD mice, where the profile more closely resembles a chronic, persistent, low-level inflammation.

I have recently read the paper by Bulloch and coworkers with great interest, which shows the presence of CD11c/EYFP “dendritic-like” mouse microglia in multiple stages of life. However, because the authors did not quantify their observations, it is unclear how prevalent these cells are in the brain, and/or whether these cells arose from the blood or were long-term CNS residents. Further, the authors had difficulty in co-staining these cells with CD11c antisera in tissue sections, raising a possibility that those who work with transgenics are all too aware of: expression of transgenes is often more promiscuous than expected. In our study, we demonstrated a seven- to eightfold increase in CD45+CD11b+CD11c+CD68+Ly-6C- cells (presumed “anti-inflammatory” macrophages initially immunophenotyped by Littman’s group in Geissmann et al., 2003) in our crossed mice, and immunohistochemical approaches revealed prominent vascular cuffing, where these cells appeared to be entering the brain via cerebrovessels. Regarding the questions from Joseph El Khoury and Pritam Das about the origin of these brain macrophages, we agree that the “acid test” of whether the macrophage-like cells that we see in and around cerebral vessels and β amyloid plaques arise from the periphery or from within the CNS would either be a chimeric approach or parabiosis. We moved away from the chimeric approach following recent reports in Nature Neuroscience (Ajami et al., 2007; Mildner et al., 2007) showing that the act of irradiating the mice leads to brain infiltration of monocytes/macrophages—the very dependent variable that we are interested in testing. However, we believe that 1) parabiosis of AD mice with GFP+CD11c-DNR mice or 2) chemical methods of ablating hematopoietic cells in AD mice followed by reconstitution with GFP+CD11c-DNR bone marrow containing or depleted of macrophages represent possible strategies that we are currently pursuing.

Finally, Pritam Das raises the interesting questions of the long-term consequences of inhibiting TGF-β signaling on peripheral macrophages and the effects on T cells. We did not observe increased peripheral numbers of innate immune cells (including macrophages and dendritic cells), CD4+ or CD8+ T cells, or B cells in CD11c-DNR mice alone or in Tg2576xCD11c-DNR crossed mice, suggesting that an autoimmune state was not generated and that the increased abundance of macrophages in the brains of our crossed mice was β amyloid-directed. We also quantified T cells in brains of our crossed mice versus singly transgenic animals, and detected that about 4-5 percent of brain hematopoietic cells were TcRαβ positive (presumed T cells), and they were divided about equally between CD4+ and CD8+ subsets—however, these numbers were similar amongst wild-type, CD11c-DNR, APP/PS1, and APP/PS1xCD11c-DNR mice, suggesting that neither the CD11c-DNR nor the APP/PS1 transgenes were able to modify brain entry of T cells. Finally, regarding the issue of assessing neurodegeneration, we are currently pursuing this line of investigation by quantitative synaptophysin immunohistochemistry and hope to answer this question in the near future.

References:
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007 Dec;10(12):1538-43. Abstract

Bulloch K, Miller MM, Gal-Toth J, Milner TA, Gottfried-Blackmore A, Waters EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC, Steinman RM, McEwen BS. CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain. J Comp Neurol. 2008 Jun 10;508(5):687-710. Abstract

Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003 Jul;19(1):71-82. Abstract

Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nat Neurosci. 2007 Dec 1;10(12):1544-53. Abstract

Monsonego A, Weiner HL. Immunotherapeutic approaches to Alzheimer's disease. Science. 2003 Oct 31;302(5646):834-8. Abstract

Richard KL, Filali M, Préfontaine P, Rivest S. Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J Neurosci. 2008 May 28;28(22):5784-93. Abstract

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

Town T, Nikolic V, Tan J. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2005 Oct 31;2:24. Abstract

Wyss-Coray T, Masliah E, Mallory M, McConlogue L, Johnson-Wood K, Lin C, Mucke L. Amyloidogenic role of cytokine TGF-1 in transgenic mice and in Alzheimer's disease. Nature. 1997 Oct 9;389(6651):603-6. Abstract

View all comments by Terrence Town

I am glad that the researchers studying transgenic models are finally confirming our results published in 2002 (Fiala et al., 2002), which showed transmigration of macrophages across the brain vessel wall and clearance of plaques by these large macrophages.

The migrating macrophages broke through ZO-1 tight junction barrier and aggregated around brain vessels similarly as in HIV encephalitis. This has been followed by a recent publication in PNAS (Fiala et al., 2007). The animal studies cannot resolve the crucial question: are macrophages of patients with AD different from those of control subjects? The answers for interested readers are available in our PNAS article and more current work presented at ICAD. Not only macrophages penetrate across the blood-brain barrier but also clear oligomeric amyloid-β from neurons.

References:
Fiala M, Liu QN, Sayre J, Pop V, Brahmandam V, Graves MC, Vinters HV. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier. Eur J Clin Invest. 2002 May;32(5):360-71. Abstract

Fiala M, Liu PT, Espinosa-Jeffrey A, Rosenthal MJ, Bernard G, Ringman JM, Sayre J, Zhang L, Zaghi J, Dejbakhsh S, Chiang B, Hui J, Mahanian M, Baghaee A, Hong P, Cashman J. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12849-54. Abstract

View all comments by Milan Fiala