Brain Research, 548 (1991) 163-171 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116570H
163
BRES 16570
Evidence for motility and pinocytosis in ramified microglia in tissue culture Paul L. Booth and W. Eric Thomas Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210 (U.S.A.)
(Accepted 4 December 1990) Key words: Ramified microglia; Tissue culture; Cerebral cortex; Video microscopy; Plasticity; Motility; Pinocytosis; Fluid cleansing
Ramified microglial cells were investigated in primary cultures of dissociated cerebral cortical tissue from rats. The identification of these cells was confirmed through immunohistochemical staining with 7 monoclonal antibodies selective for microglia. While there was significant variation in staining intensity with different antibodies, all stained the identified ramified cells; the antibodies OX-42 and ED1 yielded the most intense immunoreactivity. Based on distinctive morphological features, the microglia could be identified in living cultures where they were monitored using time-lapse video recording. This technique revealed extremely dynamic features of cellular plasticity and motility. Ramified microglia exhibited constant and rapid alterations in the size and shape of their cell body with an associated extension and retraction of processes; concomitantly, the cells moved about in a circumscribed area. These features of plasticity and motility were unique to this cell type, and correlated with OX-42 immunostaining. The microglia also possessed a differentially high level of pinocytotic activity; this too was correlated with OX-42 staining. From the nature of their morphological plasticity and motility, high pinocytosis, and cellular distribution, it is hypothesized that the ramified microglia specifically function as a system of fluid cleansing in normal brain tissue. INTRODUCTION Since the initial characterization of microglia by Rio-Hortega 31, much work has contributed to the recognition of these cells as a separate class within the central nervous system (CNS). The microglia were subsequently distinguished from other cell types 3'26, particularly oligodendrocytes, and Vaughn and Peters 39 provided one of the earliest electron microscopic descriptions of this third neuroglial element. While microglia are now well established as a distinct cell type in the CNS, there is still considerable debate concerning the functional role of these cells. Numerous recent studies, both in vivo and in vitro, indicate that the microglial cells are actually macrophages intrinsic to CNS tissue. Most of these studies involve a demonstration of the presence of cellular markers of the mononuclear phagocyte system or biochemical components selective for macrophage function in microglia. The microglial cells have been reported to posses Fc receptors 2s'29 and CD4 antigen 27, and to be capable of expressing CR3 receptors 15'25'28'29, vimentin (an intermediate filament protein of mesodermal cells) 16' 3o and interleukin-16'w'~a. In addition, several laboratories have reported that these ceils produce both class I and class II (Ia) major histocompatibility complex (MHC)
antigens 1'17'32'35'37. Other investigations support that microglia are derived from bone marrow cells (monocytes), activate lymphocytes through antigen presentation, and exhibit tumor cytotoxicity5'19. Finally, it has been suggested that the microglia display a mitogenic response to interleukin-34 and macrophage CSF (colony stimulating factor) 33. Thus, the microglia appear to possess multiple markers and functional properties of the monocyte/macrophage lineage. While there is increasing evidence that CNS microglial cells are indeed macrophages, most of this evidence supports the view that ramified microglia, the normal constituent of healthy adult brain tissue, are inactive or dormant cells having decreased expression of most cellular markers and functional attributes of macrophages. The ramified microglia are considered to be derived developmentally from an active macrophage form (amoeboid microglia) 8'~ and to convert into active macrophages in response to tissue injury (reactive microglial form)2'36; however, the ramified form of microglia appears to have down-regulated most macrophage properties (thus the synonymous term 'resting' microglia; reviewed in Jordan and Thomas 22 and Streit et al.34). Hence, the overall functional role of these cells as macrophages is mainly related to microglial forms other
Correspondence: W.E. Thomas, Department of Oral Biology, College of Dentistry, 305 W. 12th Ave., The Ohio State University, Columbus, OH 43210, U.S.A.
164 t h a n the r a m i f i e d cells (i.e. a m o e b o i d and r e a c t i v e ) ; p r e s e n t l y , n o direct f u n c t i o n for the r a m i f i e d f o r m of m i c r o g l i a has b e e n indicated. This l a b o r a t o r y has r e c e n t l y i d e n t i f i e d r a m i f i e d m i c r o g l i a in p r i m a r y tissue cultures of dissociated c e r e b r a l cortical cells 12 and utilized this p r e p a r a t i o n to i n v e s t i g a t e their intrinsic p r o p e r t i e s . Initial studies s u g g e s t e d that t h e s e cells possess differentially high levels of m o t i l i t y and p i n o c y t o t i c activity. F u r t h e r e v i d e n c e in s u p p o r t of t h e s e p r o p e r t i e s is p r e s e n t e d h e r e and u s e d to f o r m the basis for a h y p o t h e s i s on a direct f u n c t i o n specifically of the r a m i f i e d f o r m of microglial cells. P o r t i o n s of this w o r k h a v e b e e n p r e s e n t e d in a p r e l i m i n a r y f o r m llA3. MATERIALS AND METHODS Tissue culture "All experiments were performed on primary cultures of dissociated cerebral cortical cells obtained from fetal rats. The procedures for preparation and maintenance of these tissue cultures have previously been given in detail 2°'38, but will briefly be described here. Timed-pregnant rats were sacrificed by decapitation and usually 14-17 gestational day fetuses were removed from the uterus under sterile conditions; in variance to our previous studies, tissue was occasionally taken from older animals up to the neonate stage
so as to enhance the presence of microglia. Tissue was dissected from both cerebral hemispheres, dissociated by trituration, and cells plated at an approximate density of 5 × 104 cells/cm2 in 35-mm tissue culture dishes. Cells grew to confluence during the first week in culture and expressed mature features in the second week. All cultures usedin the present study were two weeks old or older. Such cultures contain a mixture of all major cell types present in adult rat cerebral cortex. Ramified microglia begin to appear in these cultures during the second week, and become a stable and significant population (10-20% of the total cells) during the third culture week ~2. lrnmunohistochemistry Immunohistochemical staining was performed on cortical cultures using an indirect fluorescence technique as previously described 2°' 2~. Cultures were fixed in a solution of 2% paraformaldehyde, 0.15% picric acid, and 0.1 M phosphate buffer (pH 7.4) for 30-60 min prior to staining. The monoclonal antibodies OX-35, OX-42, ED 1, ED2 (all obtained from Bioproducts for Science, Indianapolis, IN), MAC-l, MAC-3 (both from Hybritech Inc., San Diego, CA) and 2.4G2 (generously donated by Dr. B. Zwilling) were used at dilutions of 1:100; all are mouse monoclonals. These primary antibodies were localized with a rhodamine-conjugated goat antimouse secondary antibody (Boehringer Mannheim, Indianapolis, IN) diluted 1:10. All antibody incubations were performed for 1-3 h. After processing and mounting, stained cultures were viewed under epifluorescence microscopy using the appropriate wavelength parameters. Controls were performed by replacing the primary antibody in the procedure with phosphate buffer or antibodies against non-macrophage components (usually neuropeptides).
Fig. 1. Immunofluorescent staining of microglia with OX-42 antibody. A: phase-contrast photomicrograph of a culture field containing numerous microglia at different focal planes. B: fluorescence view of the same field showing OX-42 staining. Three stained microglia in the plane of focus are denoted by arrows; the corresponding cells are also indicated in A. C-F: corresponding phase and fluorescence images. respectively, of two different ramified microglia. Scale bar = 20 ~m for A and B. 10 j~m for C-F.
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Time-lapse recording Living cerebral cortical cultures were observed under phasecontrast optics on a microscope (Zeiss IM-35) equipped for photography, epifluorescence, and time-lapse video recording, Cultures were maintained at physiological conditions using a heated stage with a gas lamina flow attachment (models 5000 and 5010, Technology Inc., Whitehouse Station, NJ). Time-lapse recording was done with a video camera (Dage-MTI 65) connected to a black and white monitor (Sony PVM-122) via a time-lapse video recorder (Panasonic AG-6050), and photographs of recorded images were obtained through the use of a video printer (Sony UP-811) or by direct photography of the video monitor. Living ramified microglia were identified in culture fields under low magnification based on established features ~2, then individual cells or several microglia in close proximity were selected for study at higher power. These identified cells were continuously monitored for extended periods (2-6 h); the recording rate was reduced from the real-time speed of 60 frames/s to 1-4 s/frame.
Assessment of pinocytotic activity Pinocytosis was investigated using several soluble compounds; however, the fluorescent dye Lucifer yellow gave good results and proved most convenient. Therefore, this dye was preferentially utilized. The Lucifer yellow was prepared (at 0.3%) in minimal essential medium and applied to cultures for 30-60 min; the cultures were then observed under fluorescence microscopy in either the living or fixed state for pinocytotic ingestion. When pinocytotic labelling was combined with immunohistochemical staining for microglial markers, cultures were fixed and processed as described above for immunohistochemistry immediately after incubation with Lucifer yellow.
TABLE I
Staining of ramified microglia in cortical cultures by monoclonal antibodies Antibody
Relative intensity of staining*
OX-35 + OX-42 +++ ED1 + +(+) ED2 + MAC-1 ++ MAC-3 +(+) 2.4G2 + * Designation of symbols: + + + = relatively intense staining, + + = moderate staining, and + = weak or faint staining.
RESULTS R a m i f i e d microglial cells are d i s t i n g u i s h e d by several m o r p h o l o g i c a l features in these cultures; t h e y possess relatively small cell b o d i e s ( 5 - 1 5 /~m), thin b r a n c h i n g processes, a n d a n overall g r a n u l a r or vesicular a p p e a r ance to their somal m e m b r a n e a n d c y t o p l a s m 12. Cells with these s a m e features also e x h i b i t e d i m m u n o f l u o r e s cent staining with the O X - 4 2 a n t i b o d y (Fig. 1). This
Fig. 2. Examples of ramified microglia stained with MAC-1 (A), OX-42 (B), MAC-3 (C), and ED1 (D) antibodies. Scale bar = 20 #m for all.
166 antibody recognizes type 3 complement receptors which are a marker for macrophages and selective for microglia in brain tissue is. While the microglia displayed considerable variation in their detailed morphology, the features mentioned above were consistent and appeared specific as they reliably correlated with the OX-42 staining. To completely confirm the identified cells as microglia, immunoreactivity to a series of monoclonal antibodies possessing specificity for this cell type was investigated 14'22a7'29. Of the 7 antibodies utilized, all selectively stained these same ramified cells, but exhibited significant variation in staining intensity (see Table I). The OX-42 and ED1 consistently yielded the most intense level of staining. Some of the ineffectiveness of MAC-l, MAC-3 and 2.4G2 may reflect the fact that these antibodies were derived against mouse antigens; however, all showed some cross-reactivity in the rat cells with MAC-l, and to a lesser extent MAC-3, giving significant staining. In all cases, staining was greater than in controls. Also, in some instances, variability in the intensity of staining by the same antibody was noticed in different cultures or even different areas of the same culture; this was most likely attributable to alterations in the level or amount of antigen expression, as has been
reported for these cells 34. The cells stained by all of these antibodies exhibited common features consistent with the ramified microglia previously described in these cultures (Fig. 2). In addition to features of morphology and immunostaining properties, the microglia were also distinguished by their location and distribution. These cells were almost always situated on the upper surface of the confluent culture background layer and, similar to in vivo, appeared to be arranged in a somewhat regular array (Fig. 3). The primary basis for distinction of ramified microglia from other forms of this same cell in vivo is their characteristic morphology, and this is also the major reason for designating these identified cells in vitro as ramified microglial cells. Based on these established unique morphological features, their location and distribution, ramified microglia could be readily recognized under direct visual observation. This provided an excellent opportunity to investigate functional properties in the living cells. From the very first observations, it was noticed that the microglia possessed highly dynamic, unique aspects of morphological plasticity and motility. These events could be observed over time and were initially determined using still photography. By taking
167 photographs of the same cell at intervals of a few minutes (2-10), definite changes in morphology and location could be documented (Fig. 4). While still photography could reveal that these cellular changes had occurred, time-lapse video recording truly captured the dynamic nature of the ramified microglia. The microglia would change the shape and size of their cell body continuously; this was accompanied by and seemed in part attributable to a constant extension and retraction of cellular processes. In many cases, the cells appeared to compress or flatten down giving a noticeable increase in the size of their somata. While all of this was occurring, the cells also exhibited motility or migration as they moved around on the culture surface. All of these changes were not only constant or continuous, but also occurred on a
relatively rapid basis; as a result, an individual cell would have a completely different morphological shape with a displaced location within 30 min, and a field of microglia could present an entirely different appearance in about an h (Fig. 5). The migration of the ramified cells was not directional, but appeared more random. Each cell roved about in a small local area. These morphological alterations and movements were consistently and differentially associated with all microglia examined (> 50 individual cells) and, thus, appear to be characteristic for ramified cells. While the morphological features used to identify living microglia appeared selective and were shown to correspond with several antigenic markers, it was still sought to confirm that the cells displaying these dynamic properties were indeed
:4
Fig. 4. Morphological identification and characteristics of living microglial cells in cerebral cortical cultures. A: a low magnification view of a culture field. The cells labelled 1, 3 and 4 have been indentified as microglia, while cell 2 is a neuron. B and C: sequential presentations at higher power of the indicated cell 1 in the field shown in A; the microglial cell is to the left of a small neuron. D: higher magnification view of the neuron 2 from A. E and F: microglial cells 3 and 4, respectively, from A shown at higher magnification. Note the thin branching processes, and the granular or vesicular nature of the cytoplasm and cell surface (indicated by arrows). Overall, the microglia shown in C, E and F have moved slightly relative to their positions in A (see text). Scale bar = 30 ~tm for A, 12/lm for B and C, and 10 ~m for D-F.
168 microglia. Therefore, immunohistochemical staining was assessed in the same cells monitored via time-lapse recording. In morphologically identified cells exhibiting dynamic features on video recording, specific OX-42 staining was also observed (Fig. 6). In all instances examined, cells observed to be highly motile or dynamic exhibited OX-42 immunofluorescence. The vesicular appearance of the microglia somata suggested that they may be involved in endocytotic activity; however, it has been previously established that these ramified cells lack intrinsic phagocytic activity TM 13.23. Therefore, pinocytosis was investigated using several compounds, including horseradish peroxidase, India ink and Lucifer yellow; all were selectively and rapidly ingested by the microglia. Using Lucifer yellow, the dye could be observed inside individual cells and was concentrated in pinocytotic vesicles. Almost all microglia present in a culture appeared to be labelled, and this revealed broad fields of cells in an almost regular array (similar to that depicted in Fig. 3). To verify that the cells exhibiting pinocytosis were also the microglia, this activity was correlated with OX-42 immunohistochemistry. Lucifer yellow pinocytotically-labelled cells also possessed intense OX-42 fluorescent staining (Fig. 7). Thus, the ramified microglia in these cultures are pinocytotic and have unique dynamic properties. DISCUSSION
There is significant recent support for an overall role of microglial cells as intrinsic macrophages in brain tissue. In keeping with this role, several specific functions have been suggested for these cells. Amoeboid microglia have been proposed to eliminate unnecessary or improper neuronal projections during development 24'2s. The reactive microglia may perform antigen presentation and tumor cytotoxicity in adult brain 5't9, while both amoeboid and reactive cells appear to function in inflammatory processes 7"28. Amoeboid and reactive microglia, respectively, may also regulate gliogenesis in developing tissue 1° and glial scar formation in adult tissue after injury9 through secretion of interleukin-16't8 which is mitogenic to astrocytes. All of these specific functions, however, are related to the amoeboid and reactive cells - - active macrophage forms. For the ramified form of
Fig. 5. Time-lapse observations of a field of microglia. A: culture
field containing numerous microglial cells: 4 specific cells are
designated by n u m b e r s . B: the exact s a m e field is shown 77 min later (date and time at lower left). The 4 cells indicated in A are denoted by corresponding n u m b e r s . Scale bar = 40/am.
microglia, a direct specific function has not been proposed. In the present study, living ramified microglia have selectively been investigated in primary cultures of mixed cerebral cortical cells. The identified cells were confirmed as microglia through the use of 7 different monoclonal antibodies selective for microglial cells. The most prominent features observed for these cells were high morphoiogical plasticity, motility or migration, and a highly
Fig. 6. Correlation of dynamic properties with OX-42 immunostaining. A - D : 4 sequential frames covering 93 min from a time-lapse recording of a culture field containing n u m e r o u s microglia. Four ramified cells exhibiting morphological changes are indicated by arrows in each frame. (The upper cell of the lower 3 displays the most alterations; while in C and D, the middle one of the same three u n d e r g o e s a morphological an a ctive macrophage 11'23, i.e. reactive microglia.) E: the same culture transformation previously determined to corres p ond with " conversion " into " field after fixation, with the 4 cells indicated (arrows). F: same field after OX-42 immunostaining; the same 4 cells (arrows) exhibit intense fluorescent staining. G and H: the upper one and lower 3, respectively, stained cells at higher magnification. Scale bar = 50/~m for A - F and 20 a m for G - H .
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Fig. 7. Co-localization of Lucifer yellow pinocytosis and OX-42 immunofluorescence. A: an individual ramified microglial cell exhibiting OX-42 staining is shown from a culture preincubated with Lucifer yellow. B: Lucifer yellow fluorescence in the same cell; note the accumulation of the dye in vesicular structures. Scale bar = 15/~m for both.
efficient pinocytotic activity. The dynamic properties of
these cells serving as an overall system of fluid cleansing.
plasticity and motility, as well as pinocytosis, were shown to correlate with immunostaining using the monoclonal antibody OX-42, a specific microglial marker. In addition, in other studies not shown here, plasticity and motility were found to concur with pinocytosis in individual cells. This provides an image of the ramified microglia as a cell constantly engaged in rapid morphological alterations, with the extension and retraction of processes, in conjunction with pinocytotic activity, Microglial motility was marked by migration about in a small local area, supporting in combination with the above features a function of fluid cleansing in its
Thus, as an extension of their previously proposed scavenger role, it is hypothesized here that the ramified microglia specifically function as a system of fluid exchange and cleansing. While additional work is necessary to verify the presence of the properties described here in the corresponding cells in vivo, this hypothesized function could potentially contribute to the integral operation of normal brain tissue through the containment of diffusableneurotransmitter/neuromodulatorcompounds.
microenvironment. Finally, the regular distribution of ramified cells in culture, as in vivo, is consistent with REFERENCES 1 Akiyama, H., Itagaki, S, and McGeer, EL., Major histocompatibility complex antigen expression on rat microglia following epidural kainic acid lesions, J. Neurosci. Res., 20 ( 1 9 8 8 ) 147-157. 2 Brierley, J.B. and Brown, A.W., The origin of lipid phagocytes in the central nervous system: I. The intrinsic microglia, J. Comp. Neurol., 211 (1982) 397-406.
Acknowledgements. Gratitude is expressedto Dr. EL. Jordan for critical review of the manuscript, and to Ms. D. Henry and Ms J. Sonceau for assistance in its preparation. This work was supported by a Kiingenstein Fellowship in the Neurosciences (W.E.T.) and the OSU College of Dentistry.
3 Cammermeyer, J., Morphologic distinctions between oligodendrocytes and microglia cells in the rabbit cerebral cortex, Am. J. Anat., 118 (1966) 227-248. 4 Frei, K., Bodmer, S., Schwerdel, C. and Fontana, A., Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages, J. Immunol.' 137 (t986) 35213527. 5 Frei, K,, Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A., Antigen presentation and tumor cytotoxicity
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by interferon-y-treated microglial cells, Eur. J. Immunol., 17 (1987) 1271-1278. Gebicke-Haerter, EJ., Bauer, J., Schobert, A. and Northoff, H., Lipopolysaccaride-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells, J. Neurosci., 9 (1989) 183-194. Giulian, D., Ameboid microglia as effectors of inflammation in the central nervous system, J. Neurosci. Res., 18 (1987) 155-171. Giulian, D. and Baker, T.J., Characterization of amoeboid microglia isolated from developing mammalian brain, J. Neurosci., 6 (1986) 2163-2178. Giulian, D. and Lachman, L.B., Interleukin-1 stimulates astroglial proliferation after brain injury, Science, 228 (1985) 497499. Giulian, D., Young, D.G., Woodward, J., Brown, D.C. and Lachman, L.B., Interleukin-1 is an astroglial growth factor in the developing brain, J. Neurosci., 8 (1988) 709-714. Glenn, J.A., Jordan, EL., Booth, EL. and Thomas, W.E., Studies of the dynamic properties of microglial cells by timelapse video microscopy, Soc. Neurosci. Abs., 15 (1989) 512. Glenn, J.A., Jordan, EL. and Thomas, W.E., Further studies on the identification of microglia in mixed brain cell cultures, Brain Res. Bull., 22 (1989) 1049-1052. Glenn, J.A., Ward, S.A., Jordan, EL. and Thomas, W.E., Motility and pinocytotic activity as intrinsic properties of ramified microglia, J. Dent. Res., 69 (1990) 148. Graeber, M.B., Banati, R.B., Streit, W.J. and Kreutzberg, G.W., Immunophenotypic characterization of rat brain macrophages in culture, Neurosci. Lea., 103 (1989) 241-246. Graeber, M.B., Streit, W.J. and Kreutzberg, G.W., Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells, J. Neurosci. Res., 21 (1988) 18-24. Graeber, M.B., Streit, W.J. ,and Kreutzberg, G.W., The microglial cytoskeleton: vimentin is localized within activated cells in situ, J. Neurocytol., 17 (1988) 573-580. Hayes, G.M., Woodroofe, M.N. and Cuzner, M.L., Microglia are the major cell type expressing MHC class II in human white matter, J. Neurol. Sci:, 80 (1987) 25-37. Heiter, E., Ayala, J., Denefle, P., Bousseau, A., Rouget, P., Mallat, M. and Prochiantz, A., Brain macrophages synthesize interleukin-1 and interleukin-1 mRNAs in vitro, J. Neurosci. Res., 21 (1988) 391-397. Hickey, W.E and Kimura, H., Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo, Science, 239 (1988) 290-292. Jordan, EL., Rieke, G.K. and Thomas, W.E., Presence and development of ependymal cells in primary tissue cultures derived from embryonic rat cerebral cortex, Dev. Brain Res., 35 (1987) 97-110. Jordan, EL. and Thomas, W.E., Identification ofsomatostatincontaining neurons in primary cultures of rat cerebral cortex, Neurosci. Left., 77 (1987) 249-254. Jordan, F.L. and Thomas, W.E., Brain macrophages: questions of origin and interrelationship, Brain Res. Rev., 13 (1988) 165-178.
23 Jordan, EL., Wynder, H.J., Booth, P.L. and Thomas, W.E., Method for the identification of brain macrophages/phagocytic cells in vitro, J. Neurosci. Res., 26 (1990) 74-82. 24 Killackey, H.P., Glia and the elimination of transient cortical projections, Trends Neurosci., 7 (1984) 225-226. 25 Matsumoto, Y., Watabe, K. and Ikuta, E, Immunohistochemical study on neuroglia identified by the monoclonal antibody against a macrophage differentiation antigen (Mac-l), J. Neuroimmunol., 9 (1985) 379-389. 26 Mori, S. and Leblond, C.P., Identification of microglia in light and electron microscopy, J. Comp. Neurol., 135 (1969) 57-80. 27 Perry, V.H. and Gordon, S., Modulation of CD4 antigen on macrophages and microglia in rat brain, J. Exp. Med., 166 (1987) 1138-1143. 28 Perry, V.H. and Gordon, S., Macrophages and microglia in the nervous system, Trends Neurosci., 11 (1988) 273-277. 29 Perry, V.H., Hume, D.A. and Gordon, S., Immunohistochemicai localization of macrophages and microglia in the adult and developing mouse brain, Neuroscience, 15 (1985) 313-326. 30 Rieske, E., Graeber, M.B., Tetzlaff, W., Czlonkowska, A., Streit, W.J. and Kreutzberg, G.W., Microglia and microgliaderived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro, Brain Research, 492 (1989) 1-14. 31 Rio-Hortega, P., Microglia. In W. Penfield (Ed.), Cytology and Cellular Pathology of the Nervous System, Vol. 2, Hoeber, New York, 1932, pp. 481-534. 32 Sasaki, A., Levinson, S.W. and Ting, J.P.-Y., Comparison and quantitation of Ia antigen expression on cultured microglia and ameboid microglia from Lewis rat cerebral cortex: analyses and implications, J. Neuroimmunol., 25 (1989) 63-74. 33 Sawada, M., Suzumura, A., Yamamoto, H. and Marunouchi, T., Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C, Brain Research, 509 (1990) 119-124. 34 Streit, W.J., Graeber, M.B. and Kreutzberg, G.W., Functional plasticity of microglia: a review, Glia, 1 (1988) 301-307. 35 Streit, W.J., Graeber, M.B. and Kreutzberg, G.W., Peripheral nerve lesion produces increased levels of major histocompatibility complex antigen in the central nervous system, J. Neuroimmunol., 21 (1989) 117-123. 36 Streit, W.J. and Kreutzberg, G.W., Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin, J. Comp. Neurol., 268 (1988) 248-263. 37 Suzumura, A., Mezitis, S.G.E., Gonatas, N.K. and Siiberberg, D.H., MHC antigen expression on bulk isolated macrophagemicroglia from newborn mouse brain: induction of Ia antigen expression by 7-interferon, J. Neuroimmunol., 15 (1987) 263278. 38 Thomas, W.E., Synthesis of acetylcholine and 7-aminobutyric acid by dissociated cerebral cortical cells in vitro, Brain Research, 332 (1985) 79-89. 39 Vaughn, J.E. and Peters, A., A third neuroglial cell type. An electron microscopic study, J. Comp. Neurol., 133 (1968) 269-288.
163
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Evidence for motility and pinocytosis in ramified microglia in tissue culture Paul L. Booth and W. Eric Thomas Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210 (U.S.A.)
(Accepted 4 December 1990) Key words: Ramified microglia; Tissue culture; Cerebral cortex; Video microscopy; Plasticity; Motility; Pinocytosis; Fluid cleansing
Ramified microglial cells were investigated in primary cultures of dissociated cerebral cortical tissue from rats. The identification of these cells was confirmed through immunohistochemical staining with 7 monoclonal antibodies selective for microglia. While there was significant variation in staining intensity with different antibodies, all stained the identified ramified cells; the antibodies OX-42 and ED1 yielded the most intense immunoreactivity. Based on distinctive morphological features, the microglia could be identified in living cultures where they were monitored using time-lapse video recording. This technique revealed extremely dynamic features of cellular plasticity and motility. Ramified microglia exhibited constant and rapid alterations in the size and shape of their cell body with an associated extension and retraction of processes; concomitantly, the cells moved about in a circumscribed area. These features of plasticity and motility were unique to this cell type, and correlated with OX-42 immunostaining. The microglia also possessed a differentially high level of pinocytotic activity; this too was correlated with OX-42 staining. From the nature of their morphological plasticity and motility, high pinocytosis, and cellular distribution, it is hypothesized that the ramified microglia specifically function as a system of fluid cleansing in normal brain tissue. INTRODUCTION Since the initial characterization of microglia by Rio-Hortega 31, much work has contributed to the recognition of these cells as a separate class within the central nervous system (CNS). The microglia were subsequently distinguished from other cell types 3'26, particularly oligodendrocytes, and Vaughn and Peters 39 provided one of the earliest electron microscopic descriptions of this third neuroglial element. While microglia are now well established as a distinct cell type in the CNS, there is still considerable debate concerning the functional role of these cells. Numerous recent studies, both in vivo and in vitro, indicate that the microglial cells are actually macrophages intrinsic to CNS tissue. Most of these studies involve a demonstration of the presence of cellular markers of the mononuclear phagocyte system or biochemical components selective for macrophage function in microglia. The microglial cells have been reported to posses Fc receptors 2s'29 and CD4 antigen 27, and to be capable of expressing CR3 receptors 15'25'28'29, vimentin (an intermediate filament protein of mesodermal cells) 16' 3o and interleukin-16'w'~a. In addition, several laboratories have reported that these ceils produce both class I and class II (Ia) major histocompatibility complex (MHC)
antigens 1'17'32'35'37. Other investigations support that microglia are derived from bone marrow cells (monocytes), activate lymphocytes through antigen presentation, and exhibit tumor cytotoxicity5'19. Finally, it has been suggested that the microglia display a mitogenic response to interleukin-34 and macrophage CSF (colony stimulating factor) 33. Thus, the microglia appear to possess multiple markers and functional properties of the monocyte/macrophage lineage. While there is increasing evidence that CNS microglial cells are indeed macrophages, most of this evidence supports the view that ramified microglia, the normal constituent of healthy adult brain tissue, are inactive or dormant cells having decreased expression of most cellular markers and functional attributes of macrophages. The ramified microglia are considered to be derived developmentally from an active macrophage form (amoeboid microglia) 8'~ and to convert into active macrophages in response to tissue injury (reactive microglial form)2'36; however, the ramified form of microglia appears to have down-regulated most macrophage properties (thus the synonymous term 'resting' microglia; reviewed in Jordan and Thomas 22 and Streit et al.34). Hence, the overall functional role of these cells as macrophages is mainly related to microglial forms other
Correspondence: W.E. Thomas, Department of Oral Biology, College of Dentistry, 305 W. 12th Ave., The Ohio State University, Columbus, OH 43210, U.S.A.
164 t h a n the r a m i f i e d cells (i.e. a m o e b o i d and r e a c t i v e ) ; p r e s e n t l y , n o direct f u n c t i o n for the r a m i f i e d f o r m of m i c r o g l i a has b e e n indicated. This l a b o r a t o r y has r e c e n t l y i d e n t i f i e d r a m i f i e d m i c r o g l i a in p r i m a r y tissue cultures of dissociated c e r e b r a l cortical cells 12 and utilized this p r e p a r a t i o n to i n v e s t i g a t e their intrinsic p r o p e r t i e s . Initial studies s u g g e s t e d that t h e s e cells possess differentially high levels of m o t i l i t y and p i n o c y t o t i c activity. F u r t h e r e v i d e n c e in s u p p o r t of t h e s e p r o p e r t i e s is p r e s e n t e d h e r e and u s e d to f o r m the basis for a h y p o t h e s i s on a direct f u n c t i o n specifically of the r a m i f i e d f o r m of microglial cells. P o r t i o n s of this w o r k h a v e b e e n p r e s e n t e d in a p r e l i m i n a r y f o r m llA3. MATERIALS AND METHODS Tissue culture "All experiments were performed on primary cultures of dissociated cerebral cortical cells obtained from fetal rats. The procedures for preparation and maintenance of these tissue cultures have previously been given in detail 2°'38, but will briefly be described here. Timed-pregnant rats were sacrificed by decapitation and usually 14-17 gestational day fetuses were removed from the uterus under sterile conditions; in variance to our previous studies, tissue was occasionally taken from older animals up to the neonate stage
so as to enhance the presence of microglia. Tissue was dissected from both cerebral hemispheres, dissociated by trituration, and cells plated at an approximate density of 5 × 104 cells/cm2 in 35-mm tissue culture dishes. Cells grew to confluence during the first week in culture and expressed mature features in the second week. All cultures usedin the present study were two weeks old or older. Such cultures contain a mixture of all major cell types present in adult rat cerebral cortex. Ramified microglia begin to appear in these cultures during the second week, and become a stable and significant population (10-20% of the total cells) during the third culture week ~2. lrnmunohistochemistry Immunohistochemical staining was performed on cortical cultures using an indirect fluorescence technique as previously described 2°' 2~. Cultures were fixed in a solution of 2% paraformaldehyde, 0.15% picric acid, and 0.1 M phosphate buffer (pH 7.4) for 30-60 min prior to staining. The monoclonal antibodies OX-35, OX-42, ED 1, ED2 (all obtained from Bioproducts for Science, Indianapolis, IN), MAC-l, MAC-3 (both from Hybritech Inc., San Diego, CA) and 2.4G2 (generously donated by Dr. B. Zwilling) were used at dilutions of 1:100; all are mouse monoclonals. These primary antibodies were localized with a rhodamine-conjugated goat antimouse secondary antibody (Boehringer Mannheim, Indianapolis, IN) diluted 1:10. All antibody incubations were performed for 1-3 h. After processing and mounting, stained cultures were viewed under epifluorescence microscopy using the appropriate wavelength parameters. Controls were performed by replacing the primary antibody in the procedure with phosphate buffer or antibodies against non-macrophage components (usually neuropeptides).
Fig. 1. Immunofluorescent staining of microglia with OX-42 antibody. A: phase-contrast photomicrograph of a culture field containing numerous microglia at different focal planes. B: fluorescence view of the same field showing OX-42 staining. Three stained microglia in the plane of focus are denoted by arrows; the corresponding cells are also indicated in A. C-F: corresponding phase and fluorescence images. respectively, of two different ramified microglia. Scale bar = 20 ~m for A and B. 10 j~m for C-F.
165
Time-lapse recording Living cerebral cortical cultures were observed under phasecontrast optics on a microscope (Zeiss IM-35) equipped for photography, epifluorescence, and time-lapse video recording, Cultures were maintained at physiological conditions using a heated stage with a gas lamina flow attachment (models 5000 and 5010, Technology Inc., Whitehouse Station, NJ). Time-lapse recording was done with a video camera (Dage-MTI 65) connected to a black and white monitor (Sony PVM-122) via a time-lapse video recorder (Panasonic AG-6050), and photographs of recorded images were obtained through the use of a video printer (Sony UP-811) or by direct photography of the video monitor. Living ramified microglia were identified in culture fields under low magnification based on established features ~2, then individual cells or several microglia in close proximity were selected for study at higher power. These identified cells were continuously monitored for extended periods (2-6 h); the recording rate was reduced from the real-time speed of 60 frames/s to 1-4 s/frame.
Assessment of pinocytotic activity Pinocytosis was investigated using several soluble compounds; however, the fluorescent dye Lucifer yellow gave good results and proved most convenient. Therefore, this dye was preferentially utilized. The Lucifer yellow was prepared (at 0.3%) in minimal essential medium and applied to cultures for 30-60 min; the cultures were then observed under fluorescence microscopy in either the living or fixed state for pinocytotic ingestion. When pinocytotic labelling was combined with immunohistochemical staining for microglial markers, cultures were fixed and processed as described above for immunohistochemistry immediately after incubation with Lucifer yellow.
TABLE I
Staining of ramified microglia in cortical cultures by monoclonal antibodies Antibody
Relative intensity of staining*
OX-35 + OX-42 +++ ED1 + +(+) ED2 + MAC-1 ++ MAC-3 +(+) 2.4G2 + * Designation of symbols: + + + = relatively intense staining, + + = moderate staining, and + = weak or faint staining.
RESULTS R a m i f i e d microglial cells are d i s t i n g u i s h e d by several m o r p h o l o g i c a l features in these cultures; t h e y possess relatively small cell b o d i e s ( 5 - 1 5 /~m), thin b r a n c h i n g processes, a n d a n overall g r a n u l a r or vesicular a p p e a r ance to their somal m e m b r a n e a n d c y t o p l a s m 12. Cells with these s a m e features also e x h i b i t e d i m m u n o f l u o r e s cent staining with the O X - 4 2 a n t i b o d y (Fig. 1). This
Fig. 2. Examples of ramified microglia stained with MAC-1 (A), OX-42 (B), MAC-3 (C), and ED1 (D) antibodies. Scale bar = 20 #m for all.
166 antibody recognizes type 3 complement receptors which are a marker for macrophages and selective for microglia in brain tissue is. While the microglia displayed considerable variation in their detailed morphology, the features mentioned above were consistent and appeared specific as they reliably correlated with the OX-42 staining. To completely confirm the identified cells as microglia, immunoreactivity to a series of monoclonal antibodies possessing specificity for this cell type was investigated 14'22a7'29. Of the 7 antibodies utilized, all selectively stained these same ramified cells, but exhibited significant variation in staining intensity (see Table I). The OX-42 and ED1 consistently yielded the most intense level of staining. Some of the ineffectiveness of MAC-l, MAC-3 and 2.4G2 may reflect the fact that these antibodies were derived against mouse antigens; however, all showed some cross-reactivity in the rat cells with MAC-l, and to a lesser extent MAC-3, giving significant staining. In all cases, staining was greater than in controls. Also, in some instances, variability in the intensity of staining by the same antibody was noticed in different cultures or even different areas of the same culture; this was most likely attributable to alterations in the level or amount of antigen expression, as has been
reported for these cells 34. The cells stained by all of these antibodies exhibited common features consistent with the ramified microglia previously described in these cultures (Fig. 2). In addition to features of morphology and immunostaining properties, the microglia were also distinguished by their location and distribution. These cells were almost always situated on the upper surface of the confluent culture background layer and, similar to in vivo, appeared to be arranged in a somewhat regular array (Fig. 3). The primary basis for distinction of ramified microglia from other forms of this same cell in vivo is their characteristic morphology, and this is also the major reason for designating these identified cells in vitro as ramified microglial cells. Based on these established unique morphological features, their location and distribution, ramified microglia could be readily recognized under direct visual observation. This provided an excellent opportunity to investigate functional properties in the living cells. From the very first observations, it was noticed that the microglia possessed highly dynamic, unique aspects of morphological plasticity and motility. These events could be observed over time and were initially determined using still photography. By taking
167 photographs of the same cell at intervals of a few minutes (2-10), definite changes in morphology and location could be documented (Fig. 4). While still photography could reveal that these cellular changes had occurred, time-lapse video recording truly captured the dynamic nature of the ramified microglia. The microglia would change the shape and size of their cell body continuously; this was accompanied by and seemed in part attributable to a constant extension and retraction of cellular processes. In many cases, the cells appeared to compress or flatten down giving a noticeable increase in the size of their somata. While all of this was occurring, the cells also exhibited motility or migration as they moved around on the culture surface. All of these changes were not only constant or continuous, but also occurred on a
relatively rapid basis; as a result, an individual cell would have a completely different morphological shape with a displaced location within 30 min, and a field of microglia could present an entirely different appearance in about an h (Fig. 5). The migration of the ramified cells was not directional, but appeared more random. Each cell roved about in a small local area. These morphological alterations and movements were consistently and differentially associated with all microglia examined (> 50 individual cells) and, thus, appear to be characteristic for ramified cells. While the morphological features used to identify living microglia appeared selective and were shown to correspond with several antigenic markers, it was still sought to confirm that the cells displaying these dynamic properties were indeed
:4
Fig. 4. Morphological identification and characteristics of living microglial cells in cerebral cortical cultures. A: a low magnification view of a culture field. The cells labelled 1, 3 and 4 have been indentified as microglia, while cell 2 is a neuron. B and C: sequential presentations at higher power of the indicated cell 1 in the field shown in A; the microglial cell is to the left of a small neuron. D: higher magnification view of the neuron 2 from A. E and F: microglial cells 3 and 4, respectively, from A shown at higher magnification. Note the thin branching processes, and the granular or vesicular nature of the cytoplasm and cell surface (indicated by arrows). Overall, the microglia shown in C, E and F have moved slightly relative to their positions in A (see text). Scale bar = 30 ~tm for A, 12/lm for B and C, and 10 ~m for D-F.
168 microglia. Therefore, immunohistochemical staining was assessed in the same cells monitored via time-lapse recording. In morphologically identified cells exhibiting dynamic features on video recording, specific OX-42 staining was also observed (Fig. 6). In all instances examined, cells observed to be highly motile or dynamic exhibited OX-42 immunofluorescence. The vesicular appearance of the microglia somata suggested that they may be involved in endocytotic activity; however, it has been previously established that these ramified cells lack intrinsic phagocytic activity TM 13.23. Therefore, pinocytosis was investigated using several compounds, including horseradish peroxidase, India ink and Lucifer yellow; all were selectively and rapidly ingested by the microglia. Using Lucifer yellow, the dye could be observed inside individual cells and was concentrated in pinocytotic vesicles. Almost all microglia present in a culture appeared to be labelled, and this revealed broad fields of cells in an almost regular array (similar to that depicted in Fig. 3). To verify that the cells exhibiting pinocytosis were also the microglia, this activity was correlated with OX-42 immunohistochemistry. Lucifer yellow pinocytotically-labelled cells also possessed intense OX-42 fluorescent staining (Fig. 7). Thus, the ramified microglia in these cultures are pinocytotic and have unique dynamic properties. DISCUSSION
There is significant recent support for an overall role of microglial cells as intrinsic macrophages in brain tissue. In keeping with this role, several specific functions have been suggested for these cells. Amoeboid microglia have been proposed to eliminate unnecessary or improper neuronal projections during development 24'2s. The reactive microglia may perform antigen presentation and tumor cytotoxicity in adult brain 5't9, while both amoeboid and reactive cells appear to function in inflammatory processes 7"28. Amoeboid and reactive microglia, respectively, may also regulate gliogenesis in developing tissue 1° and glial scar formation in adult tissue after injury9 through secretion of interleukin-16't8 which is mitogenic to astrocytes. All of these specific functions, however, are related to the amoeboid and reactive cells - - active macrophage forms. For the ramified form of
Fig. 5. Time-lapse observations of a field of microglia. A: culture
field containing numerous microglial cells: 4 specific cells are
designated by n u m b e r s . B: the exact s a m e field is shown 77 min later (date and time at lower left). The 4 cells indicated in A are denoted by corresponding n u m b e r s . Scale bar = 40/am.
microglia, a direct specific function has not been proposed. In the present study, living ramified microglia have selectively been investigated in primary cultures of mixed cerebral cortical cells. The identified cells were confirmed as microglia through the use of 7 different monoclonal antibodies selective for microglial cells. The most prominent features observed for these cells were high morphoiogical plasticity, motility or migration, and a highly
Fig. 6. Correlation of dynamic properties with OX-42 immunostaining. A - D : 4 sequential frames covering 93 min from a time-lapse recording of a culture field containing n u m e r o u s microglia. Four ramified cells exhibiting morphological changes are indicated by arrows in each frame. (The upper cell of the lower 3 displays the most alterations; while in C and D, the middle one of the same three u n d e r g o e s a morphological an a ctive macrophage 11'23, i.e. reactive microglia.) E: the same culture transformation previously determined to corres p ond with " conversion " into " field after fixation, with the 4 cells indicated (arrows). F: same field after OX-42 immunostaining; the same 4 cells (arrows) exhibit intense fluorescent staining. G and H: the upper one and lower 3, respectively, stained cells at higher magnification. Scale bar = 50/~m for A - F and 20 a m for G - H .
170
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Fig. 7. Co-localization of Lucifer yellow pinocytosis and OX-42 immunofluorescence. A: an individual ramified microglial cell exhibiting OX-42 staining is shown from a culture preincubated with Lucifer yellow. B: Lucifer yellow fluorescence in the same cell; note the accumulation of the dye in vesicular structures. Scale bar = 15/~m for both.
efficient pinocytotic activity. The dynamic properties of
these cells serving as an overall system of fluid cleansing.
plasticity and motility, as well as pinocytosis, were shown to correlate with immunostaining using the monoclonal antibody OX-42, a specific microglial marker. In addition, in other studies not shown here, plasticity and motility were found to concur with pinocytosis in individual cells. This provides an image of the ramified microglia as a cell constantly engaged in rapid morphological alterations, with the extension and retraction of processes, in conjunction with pinocytotic activity, Microglial motility was marked by migration about in a small local area, supporting in combination with the above features a function of fluid cleansing in its
Thus, as an extension of their previously proposed scavenger role, it is hypothesized here that the ramified microglia specifically function as a system of fluid exchange and cleansing. While additional work is necessary to verify the presence of the properties described here in the corresponding cells in vivo, this hypothesized function could potentially contribute to the integral operation of normal brain tissue through the containment of diffusableneurotransmitter/neuromodulatorcompounds.
microenvironment. Finally, the regular distribution of ramified cells in culture, as in vivo, is consistent with REFERENCES 1 Akiyama, H., Itagaki, S, and McGeer, EL., Major histocompatibility complex antigen expression on rat microglia following epidural kainic acid lesions, J. Neurosci. Res., 20 ( 1 9 8 8 ) 147-157. 2 Brierley, J.B. and Brown, A.W., The origin of lipid phagocytes in the central nervous system: I. The intrinsic microglia, J. Comp. Neurol., 211 (1982) 397-406.
Acknowledgements. Gratitude is expressedto Dr. EL. Jordan for critical review of the manuscript, and to Ms. D. Henry and Ms J. Sonceau for assistance in its preparation. This work was supported by a Kiingenstein Fellowship in the Neurosciences (W.E.T.) and the OSU College of Dentistry.
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