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Circulating monocytic cells infiltrate layers of anterograde axonal degeneration where they transform into microglia

Ingo Bechmann^*,1, Jana Goldmann^*, Adam D. Kovac^*, Erik Kwidzinski^*, Eva Simbürger, Frederick Naftolin, Ulrich Dirnagl, Robert Nitsch^* and Josef Priller^,||

^* Institute of Cell Biology and Neurobiology, Charité University Hospital, Berlin, Germany;
Advanced Imaging Microscopy, Carl Zeiss Jena GmbH Division, Jena, Germany;
Department OB/GYN, Yale University, New Haven, Connecticut, USA; and
Departments of Neurology and
^|| Psychiatry, Charité University Hospital, Berlin, Germany

¹Correspondence: Center for Anatomy, Institute of Cell Biology & Neurobiology, Charité University Hospital, Berlin 10098, Germany. E-mail: ingo.bechmann{at}charite.de

SPECIFIC AIMS

Lesion-induced anterograde (Wallerian) degeneration of axons within the central nervous system (CNS) leads to rapid recruitment of cells that are immune-positive for markers of the monocyte lineage and which had been previously regarded to represent exclusively intrinsic microglia. We tested this view using entorhinal cortex lesion (ECL) to induce lamina-specific axonal degeneration in the dentate gyrus of mice grafted with GFP-transduced bone marrow and nonchimeric animals intrasplenically injected with the cell tracker CFDA, both allowing for clear-cut detection of blood-derived monocytic cells within the CNS.

PRINCIPAL FINDINGS

1. Blood-derived cells infiltrate deafferentiated layers and transform into microglia
Stereotaxic lesioning of the medial portion of the perforant path by ECL induces lamina-specific degeneration in its termination zone, the middle molecular layer (MML) of the dentate gyrus, while the inner and outer molecular layers (IML/OML) are not affected. The contralateral hippocampus is also widely unaffected by ECL. At all time points studied after lesion, GFP⁺ cells in the contralateral hemisphere were restricted to the meninges and Virchow-Robin’s spaces (Fig. 1 A, C), where perivascular macrophages are known to undergo physiological turnover. Conversely, many GFP⁺ cells of round and "amoeboid" morphologies appeared at 24 h post lesion (hpl) in the dentate gyrus and hilus of the lesioned hemisphere, as well as in the perivascular space of the vessel in the hippocampal fissure (Fig. 1B ). This was observed along the full length of the longitudinal axis of the hippocampus. At 72 hpl, the density of these cells had increased, with most of them exhibiting ramified morphologies (Fig. 1D ). As anticipated, these cells stained positive for the microglia/macrophage markers IBA-1, Mac-1, and F4/80. Morphologic transformation from round to ramified was also evident around the lesion site in the entorhinal cortex (Fig. 2 A–C). At day 7 after ECL, ramified GFP⁺ cells had accumulated specifically in the denervated MML, suggesting migration to zones of degeneration upon invasion into the dentate gyrus (Fig. 2D ).

View larger version (103K): [in this window] [in a new window]   Figure 1. Invasion of GFP+ cells after ECL. A–B) 24 h post lesion: in the unlesioned hippocampus (A), GFP expression was restricted to round cells in the pia (arrow) and elongated cells in the perivascular spaces (arrowheads), which are known to undergo physiological turnover. This distribution was clearly altered by entorhinal lesion in the ipsilateral hemisphere (B). The number of cells in the pia as well as in the perivascular spaces was clearly increased and round GFP+ cells appeared to be located within the neuropil (C–D) 72 h post lesion. While the contralateral hemisphere (C) appeared to be unchanged compared with 24 h post lesion, the number of GFP+ cells was further increased in the dentate gyrus and entorhinal cortex in the lesioned hemisphere, and a morphologic transformation toward an elongated/ramified shape can be appreciated even under low power magnification. B, D) Insets (i) show cells in the framed area at higher magnification. DG: dentate gyrus. Original magnifications: x100.

View larger version (117K): [in this window] [in a new window]   Figure 2. Morphologic transformation of infiltrating cells. At the lesion site a similar morphologic transformation as shown in Fig. 1 was evident. A) At 24 h, GFP+ cells exhibited round or elongated morphologies and accumulated within and adjacent to the site of knife injury (arrowheads). B) This picture changed within 72 h post lesion in that fluorescent cells had acquired ramified morphologies in the neuropil (arrow), while cells at the lesion site still exhibited a rather "amoeboid" shape (arrowhead). C) High power magnification provides examples of the ramified cells within the neuropil adjacent to the lesion site at 72 h post lesion. D) At 7 days after ECL, ramified cells (arrows) appeared to accumulate in the middle molecular layer (MML), which was selectively denervated by performing lesions of the medial entorhinal cortex. The arrowheads point to round and elongated cells in the adjacent pia. GCL: granule cell layer; IML: inner molecular layer; OML: outer molecular layer; original magnifications: x200 (A, B); x600 (C); x400 (D).

To rule out that the observed infiltration of blood-derived cells is an artifact of irradiation/bone-marrow transplantation, a proof-of-principle experiment was established to demonstrate such infiltration in nonirradiated, nonchimeric animals. The fluorescent cell tracker CFDA was injected into the spleens of mice in order to label a large pool of leukocytes. Twenty-four h after injection, animals underwent ECL and were killed 48 h later. In hippocampal cross sections, CFDA+ cells were found to have invaded the neuropil in zones of axonal degeneration, where they ramified and stained positive for the monocyte/microglia marker Mac-1 at 48 hpl. Thus, monocyte recruitment after ECL detected in GFP chimeras is not an artifact of bone marrow transplantation/irradiation, and occurs in nonchimeric mice.

2. GFP⁺ cells do not transform into astrocytes
Previous studies suggested that bone marrow-derived elements may differentiate into astrocytes. Although we could not observe such transdifferentiation using GFP-GFAP transgenic mice in vivo and in vitro as described elsewhere, we tested whether this may occur under the pathologic conditions induced by ECL. We did not, however, detect a single GFP⁺ cell colocalizing with glial fibrillary acidic protein (GFAP) until day 7 after lesion, either at the lesion site or in the denervated hippocampus. Thus, transformation of bone marrow cells into astrocytes apparently does not occur in the course of axonal degeneration.

3. Confocal analysis reveals homing of GFP⁺ cells within zones of axonal degeneration
The lesion site in the entorhinal cortex apparently allows direct entry of leukocytes into the neuropil (Fig. 2A, B ). From there, GFP⁺ cells may migrate along the degenerating fiber tracts into the hippocampus. Their presence in the denervated hippocampus may thus be due to such migration rather than homing across the blood-brain barrier (BBB) within the deafferentiated layers. To address this issue, confocal microscopy was performed in the MML of sections where the glia limitans was demarked with GFAP. This approach unequivocally demonstrated that GFP⁺ cells migrate across the BBB, including this first layer of the neuropil. Thus, anterograde axonal degeneration provides signals which induce the expression of chemoattractant/adhesion molecules leading to homing of leukocytes along the degenerating fiber tract.

CONCLUSIONS AND SIGNIFICANCE

A rolefor leukocytes in postlesional remodeling after ECL has not long been considered, since neither lymphocytes nor monocytes were found by Fagan and Gage (1994) to invade the layers of axonal degeneration after ECL. The inflammatory response in these layers was believed to involve exclusively intrinsic glial cells. In contrast to this view, infiltration of a few CD4 T lymphocytes into regions of degeneration after ECL has been demonstrated recently by independent groups, and data suggest that their low number is a result of insufficient costimulation and/or apoptotic elimination by local expression of the death ligand CD95L. However, in most models of inflammation, the appearance of lymphocytes is preceded or accompanied by infiltration of monocytes. In fact, a recent flow cytometry study demonstrated that the presence of putative monocytes (CD45^high) in the hippocampus occurs before invasion of T cells, and MCP-1/CCL2 expressed on microglia and astrocytes was identified as a crucial signal for this leukocyte recruitment. The technical approach, however, could not address the question of whether these cells reside in the meninges and/or the perivascular spaces or cross the BBB to infiltrate the neuropil. Here, we demonstrate such invasion of mononuclear cells into the neuropil and their subsequent transformation into microglia-like cells in situ. This is in line with previous in vitro studies showing that monocytes acquire ramified morphologies under the influence of astrocytic factors including TGF-ß.

After ECL, bone marrow-derived cells were already present at 24 h post lesion (Fig. 1) . Within 72 h these leukocytes transformed into ramified cells which cannot be distinguished from microglia on the basis of their morphology. Transformation of GFP⁺ cells into astrocytes was observed neither at the lesion site nor in the denervated hippocampus. GFP⁺ cells were not restricted to the molecular layers of the regions of anterograde degeneration, where they eventually accumulated at 7 days post ECL (Fig. 2D ), and infiltrated the hilus and the granule cell layer as well as the lesion site (Figs. 1 , 2) . It thus appears that the anterograde degeneration of axons provides stimuli to induce chemoattractant/adhesion molecule expression in the deafferentiated layer and adjacent regions.

Incomplete phagocytosis of growth-inhibiting myelin in zones of axonal degeneration has been linked to the CNS failure to regenerate after axonal injury. At least after ECL, it is now clear that the limited clearance of myelin breakdown products is not due to a lack of recruitment of mononuclear cells into zones of degeneration, but rather may result from the quick phenotypic down-modulation into what appear to be "resting" microglia. Such control over macrophage activity may on the other hand be important for keeping secondary damage such as trans-synaptic apoptosis and dendritic damage to a minimum. We have demonstrated that experimental down-regulation of the microglial response after ECL protects denervated dendrites from secondary degeneration. It will now be interesting to examine whether similar effects can be observed in CCR2-deficient mice, which lack monocytic recruitment. Also, the putative involvement of infiltrating cells in myelin phagocytosis can now be established.

With a peak around day 3, there is a strong increase in the total number of microglia/macrophages in the molecular layers due to migration, proliferation, and recruitment of monocytic cells over the BBB. Subsequently, the number of cells declines toward normal levels. This may be a result of apoptotic elimination, but we could not detect glial apoptosis within the first 10 days after ECL. An alternate explanation is that cells leave the neuropil to present antigens in draining lymph nodes as shown during experimental autoimmune encephalomyelitis. Brain-infiltrating bone marrow-derived monocytic cells have been found to express the dendritic cell marker CD11c. Thus, migration of monocytic cells after brain lesion may not only involve routes into but also out of the CNS. In this context, it will also be important to learn whether recruited monocytic cells behave differently from intrinsic microglia. Studying the issue of leukocyte trafficking after axonal lesion has only just begun and eventually may reveal that blood-derived cells play critical roles in postlesional degeneration and regeneration.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic drawing. A rapid accumulation of cells immune positive for markers of the monocytic lineage such as IBA-1 or Mac-1 (mouse) and Ox-42 or GFS-IB4 (rat) has long been appreciated to occur within the first 2 days in zones of axonal degeneration. After ECL, the total number of these cells increases by 400% and several studies demonstrated that migration of intrinsic microglia as well as their proliferation contribute to this increase. We now add that infiltration of monocytic cells across the BBB contributes as a third factor to the cellular accumulation in the deafferentiated layers. These infiltrating cells transform into what appears to be microglia with regard to their morphology. They may, however, act differently from intrinsic cells—for example, with regard to phagocytosis and antigen-presentation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2599fje;

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