Published online before print November 11, 2004
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* Instituto de Biofísica da UFRJ, and
Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
2 Correspondence: Instituto de Biofísica da UFRJ, CCS, Bloco G, Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brasil. E-mail: rlinden{at}biof.ufrj.br
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Key Words: macrophage • apoptosis • zymosan • PrPc
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PrPc is a glycosylphosphatidylinostitol (GPI)-anchored glycoprotein concentrated in lipid rafts of the plasma membrane. It is expressed mainly in the central nervous system (CNS) but also in other cell types, including macrophages and microglia. There is much interest in understanding the physiological functions of PrPc, particularly to evaluate the role of the loss of PrPc function in prion diseases [5 6 7 8 ]. This protein has been associated with synaptic activity, modulation of cell death, and signal transduction [6 , 8 9 10 ]. PrPc binds a wide range of molecules [11 12 13 14 15 16 17 ]. Its localization in the plasma membrane indicates a role in cellular interactions, such as adhesion, recognition, and ligand captation.
Peripheral macrophages may participate in the transmission [18 19 20 ] or clearance of PrPSc [21 ]. Microglial cells, the mononuclear phagocytes of the CNS, may be activated to produce neurotoxic substances and inflammatory mediators in experimental prion disease and are possibly involved in phagocytosing PrPSc and/or apoptotic neurons [19 , 22 23 24 ]. The toxic effect of the prion peptide PrP106–126 requires the presence of microglia, which are activated to release reactive oxygen species (ROS). Conversely, PrP106–126 reduces neuronal resistance to oxidative stress [5 , 25 ]. PrP106–126 also enhances phagocytosis by microglial cells [26 ] and chemotaxis of monocytes [27 ]. Thus, mononuclear phagocytes appear to be associated with the PrPc and with prion diseases in many ways.
Phagocytosis is a complex process, important for immune responses against pathogens and resolution of inflammation. It is also involved in the elimination of apoptotic cells during embryogenesis and homeostasis. Intense phagocytosis is associated with production of ROS and tissue injury [28 ]. Once triggered, phagocytosis must be restricted to the required sites, controlled, and eventually finished. A plethora of molecules modulates phagocytosis positively [29 30 31 32 33 ] or negatively [34 35 36 37 ] and is presumably involved in the achievement of an efficient and controlled response.
We examined the role of PrPc in phagocytic responses and found that PrPc negatively modulates phagocytosis. This is a novel activity described for PrPc, and it may have important consequences for normal macrophage functions as well as for the pathogenesis of prion diseases.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice
We used 3- to 4-month-old male PrPc null mice (PrP0/0), produced in a C57Bl/6J/129/sv(ev) background, or wild-type mice (PrP+/+), descendants of an F1 generation produced by interbreeding C57Bl/6J and 129/sv(ev), healthy and with normal weight [38
]. In addition, one experiment was replicated in PrP0/0 mice back-crossed from an original 129/Ola background for several generations into the C57/Bl/10 background, as well as corresponding pure genotype controls. The latter animals were kindly provided by Dr. Bruce Chesebro (Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT).
Cell culture and induction of apoptosis
Murine retinal cells, peritoneal leukocytes, and thymocytes were used as sources of apoptotic cells. Explants of PrP+/+ mice retinae were prepared as described [39
]. Briefly, mice pups at postnatal day 2 (P2), P5, and P10 were killed instantaneously by decapitation, and the retinae were dissected with fine forceps. Fragments of 
1 mm side were maintained in culture for 24 h in BME with 20 mM HEPES and 5% FCS at 37°C in 5% CO2 under orbital shaking at 80–90 rpm. Apoptosis of proliferating cells was induced with 2 µM etoposide in explants of P2 mice. Apoptosis of photoreceptors was induced in explants of P5 or P10 mice with 10 nM thapsigargin. Explants were maintained with these drugs during the entire time in culture.
Mouse leukocytes were harvested from the peritoneal cavity of 3- to 4-month-old male PrP+/+ mice with cold calcium and magnesium-free balanced salt saline solution (CMF-BSS), resuspended in RPMI 10% FCS, and cultured at 37°C in 5% CO2 for 2 h. Then, the nonadherent leukocytes were transferred to another culture flask and cultured for 24 h, after which the cells showed signs of apoptosis, detected by labeling with acridine orange/ethidium bromide as described below.
Thymuses were removed from 1- to 2-month-old female PrP+/+ mice and dissociated in FCS. After collecting cells by centrifugation and resuspending in 17 mM Tris, 140 mM NH4Cl, pH 7.2, to lyse erythrocytes, thymocytes were washed and resuspended at 106 cells/ml in RPMI-1641 medium supplemented with 10% FCS. Apoptosis was induced in thymocytes by 100 µg/ml hydrocortisone sulfate at 37°C in 5% CO2 for 6 h.
For all cell types, viability was assessed by double-labeling with a mixture of acridine orange (2 µg/ml) and ethidium bromide (2 µg/ml). Apoptosis equaled or exceeded 60%, and necrosis was <5%.
In vitro phagocytosis assay
Resident peritoneal cells were harvested from PrP0/0 and PrP+/+ mice and plated on glass coverslips at a density of 2–4 x 105 cells/well in 24-well plates for 2 h in RPMI 10% FCS. Then, the culture was rinsed 3x with CMF-BSS and incubated for 24 h with RPMI 10% FCS. Macrophages of both genotypes showed similar cell morphology and density, which was a prerequisite to resume the experiments. Macrophages were rinsed 3x with cold CMF-BSS before adding apoptotic cells.
Cells from retinal explants were dissociated with 0.025% trypsin in CMF-BSS. All cell types were added to macrophage monolayers in RPMI 1640 supplemented with 10% FCS for 1 h, after which monolayers were washed 3x with cold CMF saline, fixed with 4% paraformaldehyde in phosphate buffer 0.1 M, pH 7.4, labeled with DAPI, and examined in a Zeiss Axiophot epifluorescence microscope.
In each experiment, a total of at least 300 macrophages from triplicate or duplicate wells was counted. Results were expressed as rate of phagocytosis, that is the fraction of macrophages positive for apoptotic nuclei. The data from various experiments were pooled by taking the rate of phagocytosis in PrP+/+ macrophages in control conditions as 100%. The values taken as 100% in each experiment are indicated in the figure legends.
PI-PLC treatment of macrophages
The medium on coverslips containing macrophages was aspirated and replaced with 300 µl fresh RPMI 10% FCS containing 0.5 U PI-PLC in vehicle (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 60% glycerol) or vehicle alone. After 1 h at 37°C, the coverslips were washed 3x with CMF saline and used immediately in phagocytosis assays.
In situ phagocytosis assay
Phagocytosis was assessed in situ in retinal explants from either genotype, based on previous descriptions of diffuse in situ nick-end labeling (ISNEL) of fragmented DNA originally from apoptotic cells in phagocytic cells within the developing retina [40
]. Retinal explants from mouse pups at P2 were maintained for 24 h in culture medium without FCS. The fixed tissue was infiltrated with 20% sucrose and embedded in optical cutting temperature compound tissue-embedding medium (Tissue-Tek). Transverse sections were cut at 10 µm in a cryostat, and ISNEL was performed with the Apoptag® Plus fluorescein in situ apoptosis detection kit, according to the manufacturer’s instructions and coverslipped with Fluoromount-G.
Apoptotic nuclei were recognized by their dense and brightly labeled, condensed profiles, and cells with phagocytic activity were recognized by their diffuse labeling and elongated morphology. Both types of profiles were counted in fields of 0.013 mm2 using a Zeiss Axiophot microscope. In some experiments, the sections were examined with a Zeiss LSM 510 META confocal microscope. Counts of apoptotic nuclei were made in three fields from each of three explants of each genotype and expressed as ISNEL-positive apoptotic nuclei/mm2. Counts of diffusely labeled cells were made in the whole extension of six explants of each genotype and expressed as ISNEL-positive, diffusely labeled cells/mm2.
Induction of peritonitis in vivo
Three- to 4-month-old PrP+/+ and PrP0/0 male mice were used. Four to five mice were used for each experimental group. Peritonitis was induced by an injection of 2 mg zymosan in 0.3 ml 0.1 M phosphate-buffered saline (PBS), pH 7.4, and control mice received the same volume of vehicle. Six hours later, animals were killed with CO2, and the peritoneal cavities were washed with 3 ml PBS containing 10 mM EDTA. Aliquots of the lavage fluids were stained in Turk’s solution (0.01% crystal violet in 3% acetic acid), and total leukocytes were counted using a Neubauer hemocytometer under a light microscope (Olympus B061). Differential counting of leukocytes was made in cytospin preparations (350 rpm, 5 min), stained with May-Grunwald and Giemsa. Data are reported as 106 leukocytes/cavity. Phagocytic activity was expressed as the rate of phagocytosis (percent leukocytes associated with three or more zymosan particles). Protein content of cell-free lavage fluids was determined by the Folin technique. Readings were made at 570 nm wavelength in a plate-reader, and absorbance values were converted to mg/ml after comparison with a standard curve for bovine serum albumin (0–5 mg/ml).
Statistical tests
Data were expressed as mean ± SEM and analyzed by one-way or two-way ANOVA using Duncan’s multiple range test with SpssPC software. The number of experiments and replicates is indicated in the legend of each figure.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culturing retinal explants in vitro in the presence of FCS leads to apoptosis of ganglion cells as a result of axotomy in explants from P2, P5, and P10 mice and also in the inner nuclear layer of explants from P5 and P10 mice. In addition, the topoisomerase II inhibitor etoposide induces the death of proliferating cells. In preliminary experiments, apoptotic cells from explants of the retina of P2, P5, and P10 PrP+/+ mice maintained in control medium as well as apoptotic cells from P2 explants treated with etoposide were offered to macrophages. In all these experiments, PrP0/0 macrophage cultures showed a 50–75% higher rate of phagocytosis in vitro compared with PrP+/+ macrophages (data not shown). Thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, induces massive death of photoreceptor cells and was used in explants from P5 mice. Macrophages from PrP0/0 mice showed a significantly increased rate of phagocytosis compared with macrophages from PrP+/+ mice (Fig. 1A ).
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Figure 1. Rate of phagocytosis of apoptotic cells is higher in cultures of PrP0/0 macrophages compared with wild type. Wild-type (open bars) and PrP0/0 (solid bars) macrophages were tested for phagocytosis of apoptotic cells from explants of the retina of P5 mice, treated with thapsigargin (A). The histogram represents a pool of four experiments with macrophages plated in triplicates. (B) Wild-type (open bars) and PrP0/0 (solid bars) macrophages were tested for phagocytosis of peritoneal leukocytes. Phagocytosis was assayed without peritoneal leukocytes, with fresh nonapoptotic peritoneal leukocytes, and with apoptotic peritoneal leukocytes. The graph represents a pool of four experiments with macrophages plated in triplicates. (C) Phagocytosis was assayed without or with apoptotic thymocytes. The graph represents a pool of six experiments with macrophages plated in duplicates or triplicates. (D) The same experiment as in C was replicated with macrophages from wild-type and PrP0/0 mice from a pure C57/BL/10 background. The graph represents a pool of two experiments with macrophages plated in tri- or quadruplicates. In this and the following figures, data are shown as mean ± SEM, and the rate of phagocytosis of wild-type macrophages is taken as 100%. The reference rates of phagocytosis in wild type are (A) 39.9 ± 8.2, (B) 5.8 ± 1.7, (C) 35.0 ± 7.0, and (D) 15.4 ± 2.1. * = P < 0.05; ** = P < 0.01.
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Figure 2. Photomicrographs of macrophage cultures. Representative fields are shown of macrophages from PrP+/+ and PrP0/0 mice under epifluorescence after staining with DAPI (A, C) and the corresponding phase-contrast photomicrographs (B, D). Macrophage nuclei are indicated with arrows, and phagocytosed DAPI-stained apoptotic nuclei are indicated with arrowheads. Notice the higher rate of phagocytosis by PrP0/0 macrophages. Original bars = 50 µm.
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To further test whether the differences between knockout and wild-type macrophages were related to PrPc, we examined the effect of acute depletion of PrPc from wild-type macrophages, using PI-PLC, an enzyme that cleaves GPI-anchored proteins from the cell surface. PrP+/+ macrophages pretreated with PI-PLC showed a higher rate of phagocytosis than untreated PrP+/+ macrophages, reaching a similar level as PrP0/0 macrophages (Fig. 3 ). Depletion of PrPc and Thy-1 as a control was verified by cytofluorometry. Thus, the depletion of GPI-anchored proteins from PrP+/+ macrophage surface had the same effect on phagocytosis as the chronic absence of PrPc in PrP0/0 macrophages. Notwithstanding the nonspecific effects of PI-PLC (see Discussion), the data are consistent with the hypothesis that PrPc down-regulates phagocytosis.
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Figure 3. Depletion of GPI-anchored proteins from PrP+/+ macrophage enhances phagocytosis of apoptotic thymocytes. Wild-type (open bars) and PrP0/0 (solid bars) macrophages were tested for phagocytosis after treatment with PI-PLC or vehicle alone. The graph shows a pool of two experiments with macrophages plated in triplicates. The reference rate of phagocytosis in wild type is 13.6 ± 1.4.
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Retinal explants from P2 mice of PrP+/+ or PrP0/0 genotype were cultured without serum for 24 h, a condition in which some retinal cells within the neuroblastic (proliferative) layer undergo apoptosis. Apoptotic cells were detected in sections labeled by ISNEL by their dense and globular labeling (Fig. 4A ). Elongated cells with diffuse labeling and radial distribution were also detected and interpreted as phagocytic cells (Fig. 4A) , as many of these cells also contained apoptotic nuclei that had probably been ingested (Fig. 4A) . The diffusely labeled cells were recognized as Müller glial cells, according to the elongated transretinal morphology and the phagocytic activity described for these cells [46 47 48 49 50 51 52 53 ], although it cannot be excluded that some profiles pertain to neuroblasts. Both are, nonetheless, derived from neuroectoderm and as such, functionally distinct from mononuclear phagocytes. Estimates of the density of profiles with either pattern of labeling showed that diffuse ISNEL-positive profles were approximately 3x as frequent in retinal explants of PrP0/0 mice than in wild type (Fig. 4B) . This difference cannot be attributed to an increased number of apoptotic cells, which was similar in retinal explants of either genotype (Fig. 4C ; see also Fig. 8D, and 8E, in ref. [17 ]).
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Figure 4. In situ phagocytic activity is higher in retinal explants from PrP0/0 than from PrP+/+ mice. (A) Photomicrographs of transverse sections of explants from the retina of PrP+/+ (upper) and PrP0/0 (PrP–/–; lower) mice, labeled with the ISNEL technique (left, tridimensional reconstructions of confocal sections) or under differential interference contrast (DIC; right). Diffuse ISNEL is found in elongated cells with the morphology of Muller glia (arrows), and compact labeling is found in globular pyknotic nuclei (arrowheads). Original bars = 20 µm. (B, C) Densities of profiles with diffuse (B) or compact (C) labeling in sections of explants from wild-type (open bars) and PrP0/0 (solid bars) retinal explants. Counts were made in three fields from each of three explants of each genotype and are expressed as ISNEL-positive apoptotic nuclei/mm2. TUNEL, Deoxyuridine triphosphate nick-end labeling.
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Figure 5. Recruitment of leukocytes in response to zymosan in PrP+/+ and PrP0/0 mice. Adult wild-type (open bars) and PrP0/0 (solid bars) mice were injected intraperitoneally (i.p.) with saline (n=4 mice of each genotype) or zymosan (n=5 mice of each genotype). Peritoneal lavages were done 6 h after the injections. (A) Total numbers of harvested leukocytes. (B) Harvested leukocytes were distinguished as mononuclear leukocytes (solid bars), neutrophils (hatched bars), and eosinophils (open bars) in cytospin preparations.
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Figure 6. Association of leukocytes and zymosan is more prominent in PrP0/0 than in wild-type mice. Photomicrographs of peritoneal leukocytes from PrP+/+ and PrP0/0 mice 6 h after injection of saline or zymosan. Mononuclear leukocytes (open arrow), neutrophils (solid arrow), and zymosan (arrowhead). Original bar = 10 µm. The graph shows that phagocytosis in vivo by PrP0/0 macrophages (solid bar) is more efficient as compared with macrophages from wild type (open bar). Rate of phagocytosis in this case is the percentage of macrophages with three or more zymosan particles. The reference rate of phagocytosis in wild type is 16.0 ± 3.8.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PrPc is a GPI-anchored surface protein. To test whether the effect observed was a result of alterations in knockout mice other than the absence of PrPc, we replicated the experiment of in vitro phagocytosis of apoptotic thymocytes in two distinct strains of knockout mice. The results for a C57/129 mixed background as well as those for pure C57/Bl/10 background were similar, indicating that the distinct rates of phagocytosis cannot be attributed to spurious variations as a result of mixed genetic background. In addition, we depleted the surface of wild-type macrophages of GPI-anchored proteins using the enzyme PI-PLC. Acute depletion of GPI-anchored proteins from wild-type macrophages made these cells as efficient as knockout macrophages. Other GPI-anchored proteins, such as CD14 and Fc receptor for immunoglobulin G (Fc
R)IIIB, may participate in phagocytic responses. However, depletion of CD14 or FcRIIIB would be expected to reduce phagocytic activity [55
, 56
], and the latter is found only in neutrophils. Furthermore, although PI-PLC is not specific for PrPc, effects independent of PrPc expression should be similar in wild-type and PrP0/0 cells, which was not the case. Taken together, these results support the hypothesis that PrPc modulates phagocytosis negatively.
Phagocytosis was also examined in the retina in situ. In agreement with the in vitro assays, we found evidence for greater phagocytic activity, probably by Müller cells, in retinae of knockout mice compared with wild type. This result supports the hypothesis that phagocytic activity is modulated by PrPc in physiological circumstances and in cells of neuroectodermal origin as well as in professional phagocytes.
After i.p. injection of zymosan, phagocytosis in PrP0/0 mice was more efficient than in wild type. In addition, PrP0/0 mice recruited more monocytes and less neutrophils than wild type. Similar to phagocytosis, leukocyte recruitment can also be regulated by GPI-anchored proteins, such as the receptor urokinase-type plasminogen activator and GPI-80 [57 58 59 60 61 ]. However, modulation of leukocyte recruitment by PrPc has not been described previously. The only study linking PrPc with leukocyte chemotaxis reports that the PrP106–126 peptide is chemotactic for monoytes in vitro via N-formyl-peptide receptor-like-1 [27 ]. Preliminary experiments showed no difference in the expression of the monocyte chemoattractant protein-1 chemokine between the two genotypes following injections of zymosan (data not shown). It is likely that a comprehensive cyto- and chemokine profile will be necessary to trace the mechanisms underlying the distinct inflammatory responses of wild-type and PrPc null mice.
Diminished or augmented phagocytosis can produce undesirable responses. Phagocytosis of opsonized particles is associated with the production of oxygen free radicals and lysosomal enzymes, both with destructive potential [62
, 63
], and therefore, must be restricted to inflammation sites. Circulating monocytes should have a limited phagocytic capacity, only to be enhanced upon migration into inflamed sites [35
]. It has been argued that the limited phagocytosis of apoptotic lymphocytes by alveolar macrophages, when compared with peritoneal macrophages, helps preserve lung tissue from inflammatory destruction [64
]. Whereas most modulators of phagocytosis described to date have positive effects, PrPc adds to a growing list of negative modulators, such as the tyrosine kinase Fgr and the immunoreceptor tyrosine-based inhibitory motif domain-containing receptor signal-regulatory protein 
[35
, 65
66
67
], which may be essential to balance the threshold of phagocytic responses.
Similar to PrPc, many engulfment receptors, such as CD36, CD44, SR-BI, CD14, and FcRIIIB, are present in lipid rafts [68
], where they may interact. It is possible that ligand binding to GPI-anchored proteins modifies the threshold for cellular activation, instead of acting as a primary signal. An alternative role of GPI-anchored proteins in phagocytosis is to relocate receptors to lipid rafts, such as described for the nonopsonic phagocytosis of Mycobacterium kansasii, which is mediated by CR3 associated to GPI-anchored proteins [69
].
The main phagocyte within the CNS is microglia, derived from peripheral monocytes [70 , 71 ]. These cells invade the CNS during development and participate in the clearance of neurons undergoing programmed cell death [50 , 71 72 73 74 ]. In addition, perivascular macrophages, as well as pericytes in brain capillaries, are also phagocytic [75 , 76 ]. In the adult CNS, microglia is quiescent and can be activated by pathogen invasion, ischemia, and inflammatory responses [70 , 77 ]. Inflammation in the CNS is limited [63 ], and activation of microglia must be curtailed, as these cells produce cytotoxic and inflammatory mediators. The ability to down-regulate phagocytosis and possibly other activities of activated microglia would be an advantage in a tissue rich in PrPc such as the CNS.
Negative modulation of phagocytosis can also have implications in prion diseases, which are accompanied by activation of microglia and production of cytokines and ROS [19 ]. Conversion of PrPc into PrPSc and the ensuing depletion of PrPc from microglia may lead to an activated state, including increased phagocytic activity. In vitro cultures of microglia derived from wild-type mice seem to be more activated than PrP0/0 microglia, because of augmented production of superoxide after stimulation with lipopolysaccharide or concanavalin A [78 ] and PrP106–126 [25 ] and nitrite after stimulation with PrP106–126 [25 ], which would appear to contradict our results. However, the negative modulation of phagocytosis by superoxide [79 ] and the superoxide derivative H2O2 (refs. [80 81 82 83 ], but see ref. [84 ]) as well as nitric oxide [85 86 87 ] and its derivative nitrite [88 ] are, indeed, consistent with the observation of higher phagocytic activity of PrP0/0 macrophages. It would be of interest to study the phagocytic activity of microglia as well as the production of H2O2 of peritoneal macrophages derived from PrP0/0 mice.
The presence of PrPc affects the activation of microglia and certain leukocytes [25 , 78 , 89 90 91 ]. The distinct responses of phagocytic activity and leukocyte recruitment between PrP0/0 and wild-type mice may therefore reflect a differential profile of cytokine production. A systematic study of the cytokine profiles of PrP0/0 and wild-type mice is under way and may help explain the distinct behavior of macrophages of either genotype. It may be of significance that the high phagocytic activity as well as the low neutrophil recruitment observed in the PrP0/0 mouse are similar to the atypical inflammatory behavior of microglia in experimentally induced prion disease [92 ]. An intriguing possibility is that microglia depleted of PrPc in the course of the disease may acquire a phenotype similar to the PrP0/0 macrophages described here.
In conclusion, a regulatory activity of phagocytosis by PrPc is here described for the first time in vitro, in situ, and in vivo. This activity may be relevant for physiological and pathological processes related to normal neural development and prion diseases.
Received November 3, 2003; revised September 20, 2004; accepted October 15, 2004.
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