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Regular Article |
From the Department of Neurology,*
Universitätsklinikum Münster, Münster, Germany; the
Department of Pathology,
Dartmouth-Hitchcock
Medical Center, Dartmouth Medical School, Lebanon, New Hampshire; and
the Department of Neurochemistry,
National
Institute of Neuroscience, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endoneurial macrophages are not a homogenous cell population.6 In addition to hematogenous macrophages entering the nerve in large numbers during disease, a population of resident endoneurial macrophages exists in the normal peripheral nerve that accounts for up to 9% of the entire endoneurial cell population.6-8 This endoneurial location makes them key candidates for an early response to disease similar to the enigmatic role of microglial cells of the central nervous system.9 However, the biological role of resident endoneurial macrophages during disease in vivo is essentially unknown as there are no existing cellular markers that may discriminate them from infiltrating hematogenous macrophages. Early studies using nerve explants into the peritoneal cavity that were contained in Millipore chambers not allowing access of peritoneal macrophages, very little or no phagocytosis by resident endoneurial macrophages was found, and nonresident macrophages were required for myelin removal.10,11 However, experiments in peripheral nerve organ cultures without added macrophages suggested that resident endoneurial macrophages may phagocytose myelin to a limited degree and increase in number.12 Their possible counterparts in the central nervous system, the microglial cells, respond extremely rapidly to a wide variety of pathological stimuli and thus seem to be the primary local cells involved in immunosurveillance of the brain.9 It may thus be hypothesized that resident endoneurial macrophages provide a functionally significant contribution to the macrophage response during peripheral nerve disorders, and the characterization and study of functional properties of resident endoneurial macrophages could provide important clues to our understanding of peripheral nerve disease.
One possibility to discriminate between hematogenous and resident macrophages in laboratory animals is the induction of bone marrow chimerism. Such chimeras are created by lethally irradiating recipient animals and transplanting donor bone marrow that carries a discriminating cellular marker allowing the distinction between local and bone marrow-derived cells. Radiation bone marrow chimeric rats based on different MHC haplotypes were successfully used to investigate the physiological turnover of resident endoneurial macrophages in the normal peripheral nerve.13 However, the detection of the discriminating MHC haplotype required the stimulation of experimental animals with high-dose cytokines to up-regulate MHC molecules. TK-tsa transgenic Lewis rats carry 250 to 300 copies of a functionally silent DNA construct in all cell nuclei that may be constitutively detected by in situ hybridization.14 In the present study we created radiation bone marrow chimeric rats by transplanting wild-type Lewis rat bone marrow into irradiated TK-tsa transgenic Lewis rats to identify resident endoneurial macrophages and investigate their role in a model of Wallerian degeneration. In such animals, all resident tissue cells carry the TK-tsa transgene whereas bone marrow-derived blood cells do not. Co-localization of the transgene with macrophage markers thus unequivocally identifies transgene-positive endoneurial macrophages as resident endoneurial macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TK-tsa transgenic Lewis rats14
and wild-type Lewis
rats were obtained from BRL, Füllinsdorf, Switzerland, and
Charles River, Sulzfeld, Germany, respectively. Bone marrow chimeric
rats were created as described previously.15,16
Recipient
TK-tsa transgenic Lewis rats were lethally irradiated with 1000 rads
using a cobalt 
radiation source. Donor bone marrow cells were
prepared by extruding bone marrow from the long bones of wild-type
Lewis rats using a syringe. Bone marrow cells were then suspended in
phosphate-buffered saline, filtered through a 70-µm nylon mesh,
washed, and centrifuged at 1200 rpm twice for 10 minutes, resuspended
in buffer, and counted. Subsequently, 
107
donor bone-marrow cells in a volume of 400 µl were injected into the
tail vein of recipient irradiated TK-tsa transgenic Lewis rats. Rats
were allowed to recover for 3 months to establish mature chimerism
before further experiments were performed. For control purposes,
chimeras were created the opposite way, ie, TK-tsa transgenic bone
marrow was transplanted into wild-type Lewis rats. Unless otherwise
stated, the experiments presented in the present article were performed
in chimeras where TK-tsa transgenic Lewis rats served as recipients and
wild-type Lewis rats as donors.
Sciatic Nerve Injury
The right sciatic nerve was exposed under deep ether anesthesia and crushed just distal to the sciatic notch in a standardized way for 10 seconds with a fine forceps. TK-tsa transgenic rats carrying Lewis rat bone marrow were sacrificed 1, 2, 3, 4, 7, 14, and 28 days after crush injury. There were 21 rats in the experimental group, three animals for each time point examined. A total of 40 control rats comprising Lewis rat chimeras carrying TK-tsa bone marrow (8 rats), nonchimeric TK-tsa transgenic rats (8 rats), and nonchimeric wild-type Lewis rats (24 rats) were examined without crush injury and 1, 4, 7, and 14 days after crush injury, with additional time points for wild-type Lewis rats. The control experiments were performed to exclude any influence of chimera production, the direction of chimerism and the presence of the transgene on Wallerian degeneration, and endoneurial macrophage numbers and their response to peripheral nerve injury. All animal experiments were approved by the veterinary office at the Bezirksregierung Münster, Germany.
Assessment of Cell Proliferation
Rats received 75 mg/kg of bromodeoxyuridine (BrdU; Sigma, Deisenhofen, Germany) intraperitoneally 2 hours before sacrifice to detect proliferating cells. An additional group of chimeric animals, killed 7 days after crush injury, received drinking water containing 1 mg/ml BrdU for the entire period between crush injury and sacrifice to mark all cells that had undergone proliferation during that time.
Fixation and Tissue Preparation
Rats were perfused through the left ventricle under deep ether anesthesia for 1 minute with a 6% hydroxyethyl-starch solution (HAES sterile; Fresenius, Bad Homburg, Germany) followed by 4% phosphate-buffered paraformaldehyde at pH 7.4 for 10 minutes. Relevant tissues were postfixed in the same fixative for a further 3 hours.
Methyl Methacrylate Embedding
Sciatic nerve tissue 7-mm distal from the crush site and from the contralateral control side as well as splenic tissue was embedded in methyl methacrylate as described previously.16 Polymerization was allowed for 48 hours at -20°C under vacuum. Series of transverse adjacent 0.5-µm semithin sections were cut on a Reichert-Jung ultracut ultramicrotome (Leica, Wetzlar, Germany), transferred onto coated glass slides, and dried at 35°C for 2 hours.
TK-tsa Probe for in Situ Hybridization
A plasmid containing the sequence of the TK-tsa
transgene14
was used as a template to produce
digoxigenin-labeled DNA probes by polymerase chain reaction according
to standard protocols. Four regions of the transgene were amplified by
different sets of primer pairs (Table 1)
,
yielding a probe cocktail with four labeled probes of 200 bp each.
Labeling efficiency was tested by dot-blot analysis of labeled
polymerase chain reaction products in comparison with serial dilutions
of a labeled standard sequence. All reagents used for the digoxigenin
labeling and dot-blot procedures were purchased from Roche (Mannheim,
Germany).
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In situ hybridization was performed as previously described.16 In brief, sections were cleared from methylmethacrylate by incubation in acetone and pretreated by microwave irradiation. Hybridization was performed in a humid chamber at 37°C for 14 hours in a mixture containing the labeled denatured DNA probe at 5 ng/µl, 5 mg/ml denatured salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 0.02% sodium dodecyl sulfate (all from Sigma) in 2x standard saline citrate (SSC) (1x SSC = 0.15 mol/L sodium chloride, 30 mmol/L trisodium citrate, pH 7). Before hybridization, sections and probe were denatured again at 95°C on a hot plate for 5 minutes. Washes were in 2x SSC for 3 x 20 minutes and 50% formamide/1x SSC at 50°C for 15 minutes. Hybridized probe was detected by a horseradish peroxidase-conjugated anti-digoxigenin antibody (DAKO, Hamburg, Germany), tyramide amplification, and fluorescent Cy3-streptavidin (Amersham, Braunschweig, Germany) or A594-streptavidin (Molecular Probes, Leiden, The Netherlands). Sections were counterstained with 4,6-diamidino-2-phenylindol (DAPI; Vector Laboratories, Burlingame, CA). To estimate the sensitivity and specificity of in situ hybridization for each experiment, in situ hybridization on serial sections of splenic tissue from TK-tsa transgenic and wild-type Lewis rats was always included as a control.
Immunohistochemistry and Double-Labeling Immunohistochemistry
Deplasticized tissue sections were pretreated and incubated with
primary monoclonal or polyclonal antibodies as summarized in Table 2
. Secondary anti-mouse or anti-rabbit
horseradish peroxidase-conjugated antibodies (DAKO) were used, followed
by tyramide amplification and fluorescent signal detection with
A488-streptavidin or A594-streptavidin (Molecular Probes) as described
previously.16
Negative controls were performed without
primary antibody or with an irrelevant primary antibody at comparable
concentrations. Double-labeling immunohistochemistry on single sections
using one polyclonal and one monoclonal antibody was achieved by
sequentially blocking excess biotin sites with avidin and excess avidin
sites with biotin (DAKO) as well as residual horseradish peroxidase
activity with H2O2 for 10
minutes each before starting the detection procedure for the second
primary antibody. Negative controls again included sections where one
or both primary antibodies were omitted or replaced by irrelevant
antibodies.
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Combined in situ hybridization and immunohistochemistry on single sections was done as described previously.16 In brief, sections were first hybridized as described above. The primary antibody for the immunohistochemistry procedure was co-incubated with the horseradish peroxidase-conjugated anti-DIG antibody needed to detect the hybridized in situ hybridization probe. After incubation with fluorescent streptavidin to detect the in situ hybridization signal, excessive biotin, avidin, and horseradish peroxidase was blocked as described above, followed by the immunohistochemistry protocol as described with a different fluorescent marker. Negative controls included sections where the primary antibody or the specific DNA probes were omitted.
Co-Localization of DNA and Multiple Antigens on Adjacent Sections
For this purpose, series of adjacent 0.5-µm semithin sections were prepared and processed for in situ hybridization and immunohistochemistry as needed. Most series consisted of up to eight adjacent sections of which up to four were used for in situ hybridization to increase the sensitivity of detecting transgene-positive resident cells. Individual cells were identified on each section using landmark histological features as a reference.
Image Acquisition and Processing
Sections were examined under a Leica DM fluorescence microscope and images were digitized and transferred to a PC using a Diagnostic Instruments SPOT II camera system (Visitron, München, Germany). Merging of fluorescent signals was done by Adobe Photo Shop 4.0 or the SPOT II software.
Quantitative Studies
To compare the various control groups and the experimental group of TK-tsa transgenic Lewis rats transplanted with wild-type Lewis rat bone marrow, the number of proliferating cells, the total number of endoneurial cell nuclei, and the total number of endoneurial macrophages were quantified on transverse sections of uninjured and injured sciatic nerve at selected time points. Two randomly selected fields covering approximately two thirds of the nerve area were analyzed in three different animals per group. Only Iba1-positive macrophages clearly associated with a nucleus were counted.
The degree of the physiological turnover of resident endoneurial macrophages was assessed in uninjured sciatic nerves at the mid-thigh level from chimeric TK-tsa transgenic Lewis rats transplanted with wild-type Lewis rat bone marrow. Transverse sections double-stained for Iba1 (ionized calcium-binding adaptor molecule 1)17 and the TK-tsa transgene were analyzed. Iba1-positive endoneurial macrophages in clear association with a nucleus were counted on randomly selected fields, and the percentage of cells with and without hybridization for the TK-tsa transgene was determined. Control sections from TK-tsa transgenic, nonchimeric rats were included with each in situ hybridization experiment to determine the sensitivity of in situ hybridization on single sections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sensitivity of TK-tsa in situ hybridization was studied on single sections as well as serial tissue sections from TK-tsa transgenic rats. In brain, 44% of the cell nuclei hybridized with the TK-tsa probe when one single section was analyzed. When analyzing four adjacent serial sections, 90% of cell nuclei were TK-tsa-positive on at least one of the four sections. In peripheral nerve, 31% of nuclei hybridized on single sections and 77% positive nuclei were found when four serial sections were analyzed. In wild-type rats, no specific in situ hybridization signal for the TK-tsa transgene was found.
The effectiveness of chimerism induced by bone marrow transplantation into irradiated rats was studied by in situ hybridization for the TK-tsa transgene, with results similar to those described in a previous study from our laboratory.16 In chimeras where TK-tsa transgenic rats served as recipients and wild-type rats as donors as used in all following experiments, the majority of parenchymal brain and peripheral nerve cell nuclei hybridized with the TK-tsa transgene on at least one section when four serial sections were investigated. These results were similar to those in nonchimeric TK-tsa transgenic rats used as controls. In spleen, only a few cells, mainly endothelial cells and putative stromal cells in the red pulp, were TK-tsa-positive whereas most cells within the follicles did not hybridize, indicating their derivation from bone marrow cells. In control chimeras where wild-type rats served as recipients and TK-tsa transgenic rats as bone marrow donors, opposite results were found in spleen, whereas most brain and sciatic nerve cell nuclei did not contain the transgene. There was no evidence of inflammatory autoimmune disease in any of the chimeric animals.
Wallerian Degeneration in Radiation Bone Marrow Chimeric Rats and Controls
To control for possible differences in the cellular response to
peripheral nerve injury in chimeric TK-tsa transgenic and wild-type
Lewis rats, the kinetics of axonal and myelin breakdown and local
cellular reactions were compared in normal and injured nerves.
Quantitative studies of endoneurial cell nuclei, proliferating
endoneurial cell nuclei, and endoneurial macrophages revealed no
significant difference between the four groups of animals (Tables 3 and 4)
.
Qualitative assessments of the density and breakdown of NF4-positive
axonal profiles, axonal regeneration as revealed by the occurrence of
small, thinly myelinated axonal profiles distal to the lesion, and
myelin breakdown as detected by immunohistochemistry for myelin basic
protein gave similar results in all four groups. There was also no
difference in endoneurial macrophage morphology and the frequency of
macrophages containing myelin basic protein indicating myelin
phagocytosis (data not shown). In summary, no significant difference of
selected features of Wallerian degeneration could be found between the
two types of bone marrow chimeras, TK-tsa transgenic Lewis rats and
normal wild-type Lewis rats.
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Macrophages detected by Iba1 antibody17
were
distributed throughout the endoneurium in the normal sciatic nerve
(Figure 1
; A, C, and D; Figure 4A
). In
cross-sections they appeared triangular, sometimes with tiny, slim
processes, and frequently co-localized with the TK-tsa transgene
(Figure 1, D and E)
. Some endoneurial macrophages filled the gap
between myelinated axons and seemed to attach to the myelin sheaths.
Others were located close to endothelial cells of endoneurial
capillaries. Quantitative studies revealed that 5% of the total
endoneurial cell population expressed the Iba1 macrophage antigen. The
physiological turnover of resident endoneurial macrophages with bone
marrow-derived cells in the sciatic nerve was determined in uninjured
nerves from radiation bone marrow chimeric rats matured for 3 months.
Co-localization of the Iba1 antigen with the TK-tsa transgene on
multiple adjacent sections revealed that 41% of all Iba1-positive
endoneurial macrophages were TK-tsa-positive on at least one of four
sections, whereas the others were not. Considering the sensitivity of
the in situ hybridization procedure of 77% on four adjacent
sections as shown above, a calculated number of 53% of endoneurial
macrophages were TK-tsa transgenic and thus resident endoneurial
macrophages without turnover from the blood within the 3 months since
bone marrow transplantation. We did not observe any morphological
differences between TK-tsa-positive and TK-tsa-negative resident
endoneurial macrophages. In control animals without peripheral nerve
injury, these Iba1-positive macrophages did not express the ED1 antigen
present on activated macrophages using MMA-embedded tissue.
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One day after crush injury, the first time point examined in this
study, no alterations of endoneurial macrophages in the distal nerve
stump were observed. Two days after crush injury, the cell bodies of
most Iba1-positive endoneurial macrophages began to enlarge while their
cell processes appeared retracted and thicker (Figure 1, F and H)
.
These changes were more pronounced at days 3 and 4, and their number
increased (Figure 1B)
. Co-localization with the TK-tsa transgene on
serial sections revealed that many of these activated endoneurial
macrophages carried the TK-tsa transgene and thus were resident
endoneurial cells (Figure 1, F and G)
. At later time points,
identification of such cells became increasingly difficult as
macrophage numbers rapidly increased because of influx of macrophages
from the blood.
As early as 2 days after nerve injury, occasional macrophages were
found that were highly immunoreactive for the lysosomal macrophage
antigen ED1 (Figure 1, I and J)
, a feature not found in methyl
methacrylate-embedded normal nerves. These cells always had large,
rounded cell bodies with retracted processes in contrast to the slim
appearance of quiescent endoneurial macrophages. Co-localization with
the TK-tsa transgene on the same or adjacent sections again revealed
that many of those cells contained the transgene in their genome and
thus were resident endoneurial macrophages (Figure 1, I and J)
. At 3
and 4 days, their numbers slightly increased. At all later time points,
occasional cells of this type were always found but, much like
Iba1-positive resident macrophages, they were increasingly difficult to
detect among the many hematogenous macrophages infiltrating the nerve.
However, ED1-positive resident macrophages carrying the TK-tsa
transgene were still identified at 28 days after nerve crush, the
latest time point examined.
Myelin Phagocytosis by Identified Resident Endoneurial Macrophages during Wallerian Degeneration
To further investigate features of macrophage activation in
identified resident endoneurial macrophages, we studied myelin
phagocytosis by co-localizing myelin basic protein as a marker for
myelin-containing debris with the macrophage-specific antigens Iba1 and
ED1 and the TK-tsa transgene. Two days after crush injury,
ED1-positive, TK-tsa-positive resident endoneurial macrophages were
found that contained myelin debris (Figure 2)
, indicating early myelin phagoytosis
by identified resident endoneurial macrophages. Their numbers increased
at days 3 and 4. At days 7 and 14, a large number of ED-1-positive
macrophages containing myelin debris were found, but the majority of
those macrophages were now TK-tsa-negative and thus of hematogenous
origin. At day 28, there were still many postphagocytotic, mostly
TK-tsa-negative macrophages but immunoreactivity for myelin basic
protein was lost in the debris. Phagocytosis was never found in
Iba1-positive, ED1-negative macrophages.
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To investigate whether resident endoneurial macrophages contribute
to the increase in macrophage numbers after crush injury, cell
proliferation was assessed by nuclear BrdU incorporation. Proliferating
cell profiles were found from day 2 onwards with maximum mitotic
activity on day 3. Co-localization of nuclear BrdU incorporation with
the macrophage antigen ED1 revealed many ED1-negative proliferating
cells, whereas ED1-positive macrophages were never found to proliferate
(Figure 3, A and B)
. However, when the
macrophage antigen Iba1 was co-localized with BrdU, many Iba1-positive
macrophages were found to incorporate BrdU between days 2 and 7, with
maximum mitotic activity at day 3 (Figure 3C)
. Co-localization of
Iba1-positive proliferating cells with the Schwann cell marker S-100
revealed that these cells were always S-100-negative and displayed
morphology different from Schwann cells. Co-localization with the
TK-tsa transgene on multiple serial sections revealed that many
proliferating Iba1-positive, S-100-negative macrophages carried the
transgene thus indicating that they were resident endoneurial
macrophages (Figure 3, D and E)
.
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To clarify the apparent discrepancy between our above finding of
abundant proliferation in Iba1 but not ED1-positive resident
macrophages, we first co-localized the two antigens at various time
points. Although occasional Iba1-positive, rounded and enlarged
macrophages expressed ED1 at a high level as early as 2 days after
injury, the majority of Iba1-positive macrophages revealed a much
slower increase in ED1 immunoreactivity. At day 2, usually only a few
small ED1-immunoreactive granules were found that then gradually
increased in size until, at day 28, nearly the entire cell appeared to
be filled with ED1-immunoreactive material. At the same time, the
macrophages retracted their processes as described above and became
large and rounded with much thicker processes or no processes at all
(Figure 4)
. We then fed additional
chimeric rats with BrdU continuously for 7 days after crush injury to
detect proliferating cells within that time period. In such animals we
found ED1-positive, Iba1-positive macrophages that had incorporated
BrdU into their nuclei. Additional co-localization with the TK-tsa
transgene revealed that many of these cells were TK-tsa transgenic,
resident macrophages (Figure 5)
. These
cells typically were very large rounded macrophages containing
postphagocytotic vacuoles. There were still occasional Iba1-positive
macrophages that had undergone proliferation as indicated by BrdU
incorporation but did not express the ED1 antigen and appeared in a
more ramified morphology.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings became possible through the availability of TK-tsa transgenic Lewis rats as partners for the creation of bone marrow chimerism. The functionally silent TK-tsa transgene is constitutively present in all cells of the transgenic animals and can readily be detected by in situ hybridization.14 The ease of handling, however, does depend on the use of methyl methacrylate as embedding medium.16 This resin proved to be very useful for our work, allowing highly sensitive histochemical stains with superior morphological resolution and the co-localization of DNA and multiple antigens on adjacent serial sections. One drawback is that not all transgenic cells hybridize on one single semithin section. Therefore, absence of the TK-tsa in situ hybridization signal on one section does not necessarily identify this cell as hematogenous as the transgene may be located elsewhere in the nucleus and picked up only on an adjacent serial section. The technology used in our experiments is thus an excellent tool for the study of individual cells, but quantitative studies are extremely difficult to perform and require complex corrective calculations. Studies aiming at establishing a different chimera system allowing easier quantification are underway. Another point of consideration is that resident endoneurial macrophages undergo physiological turnover from the blood that was reported to amount to 60% of all endoneurial macrophages after 3 months,13 correlating well with the calculated figure of 53% TK-tsa transgenic resident macrophages without turnover as found in our present study. Therefore, only those resident macrophages can be identified as resident in a lesion model that did not undergo turnover from the blood since bone marrow transplantation. The frequency of resident endoneurial macrophages after a lesion is thus underestimated by the rate of preceding physiological turnover.
The capacity of resident endoneurial macrophages to phagocytose myelin was previously demonstrated in organotypic sciatic nerve cultures, allowing the study of Wallerian degeneration in the absence of hematogenous macrophages.12 In this paradigm, marked phagocytic activity was demonstrated by resident macrophages, but removal of myelin was more effectively performed by peritoneal macrophages added to the cultures, as in another model of nerve explants.18 Similar results were obtained in vivo in animals depleted from peripheral macrophages by irradiation or toxic silica.18,19 It was thus concluded that the contribution of resident endoneurial macrophages to myelin phagocytosis may be minor.
Our own in vivo results now indicate that not so much the quantity but rather the time course of myelin phagocytosis may highlight the functional relevance of phagocytosis by resident endoneurial macrophages. Myelin phagocytosis was demonstrated as early as 2 days after injury. It was found to be one of the first features of macrophage activation observed and occurred several days before the influx of hematogenous macrophages that begins around day 4, consistent with previous data.20,21 Although hematogenous macrophages may be more effective in removing myelin, resident macrophages do it earlier. Phagocytosis and processing of antigen is a prerequisite for antigen presentation to T cells in inflammatory conditions. Endoneurial macrophages constitutively express MHC II molecules,22 and strong up-regulation occurs after lesions and during inflammatory neuropathy.20,22,23 Co-stimulatory B7-1 molecules are present on endoneurial macrophages during inflammatory neuropathies.3 Endoneurial macrophages are thus equipped to at least potentially present antigen, and an early phagocytotic capacity as demonstrated in our work may be a prerequisite for antigen processing. However, antigen presentation is a feature of autoimmune neuropathies and not required for the cellular cascade during Wallerian degeneration as studied in the present work. It is conceivable that early phagocytosis may be a general and nonspecific feature of resident endoneurial macrophage activation independent of the cause of nerve damage. From our present knowledge it is unlikely that early phagocytosis is needed to remove myelin because this is much better done by hematogenous macrophages.5 Rather, it may represent the alertness of resident endoneurial macrophages to pathology and the potential to present antigen.
Another feature of early activation of resident endoneurial macrophages is proliferation. This was an unexpected finding as Schwann cells are considered to be the main proliferating cell type after nerve injury. Iba1-positive proliferating cells did not co-localize with the Schwann cell marker S-100, thus excluding cross-reactivity of Iba1 with Schwann cells and proving their identity as proliferating macrophages. An increase in endoneurial macrophage numbers was also found in earlier studies using explant cultures but proliferation was not formally shown.12 However, microglial cells of the central nervous system are known to proliferate rapidly in response to injury.9,24 Using long-term BrdU supplementation for 7 days we could now identify full-blown activated ED1-positive macrophages that had incorporated BrdU and carried the TK-tsa transgene. These macrophages are phenotypically indistinguishable from hematogenous macrophages infiltrating the nerve in large numbers at this time point. However, the presence of the TK-tsa transgene unequivocally identifies them as resident macrophages, and BrdU incorporation documents preceding proliferation. It thus seems that the high numbers of macrophages in the degenerating peripheral nerve found at later stages during Wallerian degeneration are not exclusively derived from a hematogenous source. Rather, resident macrophages may provide a considerable and as yet unrecognized contribution, and both resident and hematogenous macrophages may feed the total endoneurial macrophage pool during Wallerian degeneration.
One of the most striking features is the extremely rapid response of resident endoneurial macrophages that involves changes in morphology and immunophenotype, proliferation, and phagocytosis. This pattern has remarkable similarities to the response of resident microglial cells, the local macrophage system of the brain. Microglia respond to a wide variety of stimuli with a graded response that ranges from a slight up-regulation of complement receptors to the differentiation into full-blown macrophages. In particular, microglial cells proliferate peaking around 2 and 3 days after a lesion,25 just as resident endoneurial macrophages do in our system. Early activation and proliferation of a pool of resident macrophages/microglia may thus be a general feature both in the central and peripheral nervous system. However, there are also marked differences between resident endoneurial macrophages and microglia. Resident endoneurial macrophages undergo turnover from the blood13 whereas microglial cells do not.26 Furthermore, they constitutively express the ED2 antigen22 whereas microglia do not. They thus more closely resemble the perivascular macrophages of the brain15,27 that undergo an exchange from the blood26 and also express the ED2 antigen.28 The role of perivascular macrophages of the brain during disease is primarily undetermined but antigen presentation is one of their important capabilities.15 Resident macrophages of the peripheral nervous system thus seem to represent a local macrophage population that carries features of both microglial cells and perivascular cells of the brain. However, it is presently undetermined whether the entire population of resident macrophages undergoes turnover from the blood or whether a truly local population persists. We also observed occasional cells that express the ED1 antigen very early and phagocytose, whereas others become activated more slowly, proliferate, and later begin to express the ED1 antigen. It is thus conceivable that resident endoneurial macrophages are not a homogenous cell population but rather comprise different subsets with different turnover characteristics and activation patterns.
In summary, our results document an extremely rapid response of resident endoneurial macrophages to injury that precedes the influx of blood-derived macrophages and resembles the microglial response of the brain to injury. Much like microglial cells of the brain, resident endoneurial macrophages may be primarily involved in surveillance of the peripheral nervous system and as sensors of pathological events.24 By virtue of their location and rapid response, they may act primarily as regulating cells, whereas later arriving hematogenous macrophages may take the role of effector cells of tissue destruction and repair.
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Acknowledgements |
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Footnotes |
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Supported by the Deutsche Forschungsgemeinschaft (Ki 532/3-1 and -2) and the Innovative Medizinische Forschung Program, Medical Faculty, Westfälische Wilhelms-Universität Münster.
Accepted for publication September 9, 2001.
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