Neuroscience Letters, 123 (1991) 27-31

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© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 0304394091000804 NSL 07508

Pinocytotic activity in ramified microglia Jeffery A. G l e n n , P a u l L. B o o t h a n d W. Eric T h o m a s Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210 (U.S.A.)

(Received 13 July 1990;Revisedversionreceived8 October 1990;Accepted 11 October 1990) Key words: Microglia;Brain macrophage;Cerebralcortex; Pinocytosis;OX-42 immunoreactivity;Fluid cleaning; Rat

Pinocytoticactivity was investigatedin rat cerebral cortex using the soluble tracers horseradish peroxidaseand Luciferyellow. A subpopulation of ceils selectivelyaccumulated both compounds and the labelling was mainly present in pinoeytoticvesiclesassociated with the cell body. Labelled cell bodies were small, round to oval, and distributed in an almost regular array throughout the tissue. Based on distinctivemorphologicalfeatures, some of the labelled cells could be determined as ramified microglia. This identificationwas confirmedby immunofluorescencestaining with the monoclonalantibody OX-42,which specificallyrecognizesmicroglia;OX-42 stainingconsistentlyco-localizedwith pinocytoticlabelling. The possibility that ramifiedmicroglialceilsperform a normal function of continuous fluidexchangein brain tissue is discussed.

There is increasing evidence that microglia are intrinsic macrophages in brain tissue. Included in this evidence are their possession of multiple macrophage antigenic markers [9, 10, 17, 18], the synthesis and secretion of interleukin-1 [6, 11] and interleukin-3 [7], production o f superoxide anion [3], and a significant phagocytic capacity [1, 6, 10, 18]. Additionally, the microglial cells have been suggested to be derived from monocytic blood cells [12, 16]. Finally, recent reports also indicate that microglia can express M H C class II (Ia) antigen [18, 21, 22], and are capable of antigen presentation leading to lymphocyte activation [5, 12]. Thus, the very early contention of Rio-Hortega [19] that these cells were phagocytes internal to the CNS appears accurate. While there is support for a role of the microglia as macrophages, the form of these cells normally found in adult tissue (i.e. ramified or resting microglia) appears to have down-regulated or significantly decreased the expression of most macrophage functional properties [1, 14]. The ramified microglia therefore are considered as inactive or dormant precursors which give rise to active macrophages (i.e. reactive microglia) in response to injury or infection [2, 14]. This scheme explains the overall role of microglial cells as well as the relationship of various cell forms; however, it leaves the ramified microglia without a direct constitutive function. In the absence of a requirement for active macrophages, for ramified cells, the microglial form present in normal adult tisCorrespondence: W.E. Thomas, Department of Oral Biology,College

of Dentistry, 305 W, 12thAvenue, The Ohio State University,Columbus, OH 43210, U.S.A.

sue, no direct function in brain physiology has been indicated. This laboratory has recently identified specifically ramified microglia in primary cultures of mixed cerebral cortical cells derived from fetal rats [8]. In subsequent studies, it was shown that these cells in vitro lacked phagocytic capacity, but possessed efficient pinocytocic activity and a high level of ceUular motility [23] (W.E. Thomas, in preparation). These findings led us to suggest that ramified cells may contribute to normal brain function through removal of soluble components from the extracellular fluid. In a further investigation of this hypothesis, here the demonstration o f pinocytosis selectively in ramified microglia in intact brain tissue is reported. To assess pinocytotic activity, coronal slabs or wedges (3-5 mm thick) of brain tissue were prepared. Young adult rats (Sprague-Dawley) were sacrificed by decapitation, brain tissue rapidly removed, and slabs taken from the whole cerebrum. Fresh tissue slabs were incubated for 30-60 min at 37°C in a solution of the soluble tracer horseradish peroxidase (HRP, 3.25%) or Lucifer Yellow (0.3%); the tracers were prepared in Eagle's minimal essential medium with 17 m M glucose added, and all incubations were performed at pH 7.4. After incubation, slabs were rinsed extensively in phosphate-buffered saline, pH 7.4, then fixed in a solution of 2% paraformaldehyde, 0.15% picric acid and 0.1 M phosphate buffer for 12 h at 4°C. Blocks of cortical tissue several millimeter wide were cut from each cerebral hemisphere of fixed slabs and used to preparate sections (10-40/tm). For HRP-incubated tissue, blocks were sectioned on a vibra-

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Fig. 1. Pinocytoticlabelling of cells in cerebralcortex. A: low magnificationview of fluorescentcells in Luciferyellow-incubatedtissue. B: two cells from the same fieldin A (indicatedby arrows) are shown at highermagnification.C: dark-fieldmicrographof a fieldin a sectionfrom HRP-incubated tissue. D: individual HRP-labelledcell from field in C (indicated by arrow). Higher power views (B,D) reveal the presenceof label in pinocytotic vesicles. Bar =50/lm (A,C) and 10/tm (B,D).

tome and sections mounted on glass slides. The H R P reaction was performed on these sections using a standard procedure with tetramethylbenzidine as substrate, and the slides processed for light microscopy. Lucifer yellow-incubated tissue was sucrose infiltrated and embedded; frozen tissue blocks were sectioned with a cryostat. Sections were affixed to gelatinized slides, coverslipped in glycerol and viewed under epifluorescence microscopy using the appropriate wavelength parameters. In some cases, indirect immunohistochemical staining using the monoclonal antibody OX-42 was performed on Lucifer yellow-labelled sections according to an established technique [13]. This antibody recognizes CR3 receptors in macrophages and has been shown to selectively stain microglia in brain tissue [9, 10, 20]. Sections examined for pinocytotic activity were always taken from the interior of tissue blocks. While many cells were labelled at the cut edge of the fresh tissue, some apparently by diffusion or transport, giving an irregular and dense appearance, a consistent

pattern was observed in interior regions with both Lucifer yellow and HRP. Labelling was selectively associated with a subpopulation of cells; the label was usually concentrated in cell somata which were small ( < 10/~m), round to oval, and scattered throughout the tissue (Fig. 1A,C). The labelled cells in this pattern appeared to form a somewhat regular array within the tissue. This pattern or distribution was observed in both gray and white matter, although cells of the gray matter often labelled more avidly. Such labelling appeared to correspond to pinocytotic activity since at higher magnification much of it could be seen to be confined to vesicular s t r u c t u r e s - pinocytotic vesicles (Fig. 1B,D). Based on these findings, a specific cell population in cortex displays differentially high pinocytosis. There was some variation in the intensity of labelling between individual cells, and by using less intense cells and longer photographic exposures, aspects of their overall morphology could be revealed. These cells possessed morphological features characteristic for ramified microglia (Fig. 2A-

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Fig. 2. Pinocytoticlabellingin ramifiedmicroglia.A~: individualramifiedrnicrogliawith Luciferyellowlabellingvia pinocytosisidentifiedunder epifiuorescencemicroscopy.D: exampleof a network of fiuorescentlylabelledcellularprocesses. Bar = 10 pm (A-C) and 30/zm(D).

C). Also, in some areas, a rather dense network of labelled processes, some or most apparently attributable to microglia, was revealed (Fig. 2D). No selective association of such networks with specific areas or even white versus gray matter could be detected. In a final experiment, sections from Lucifer yellowincubated tissue were subjected to fluorescence immunohistochemical staining for OX-42 antigen. This procedure selectively visualized the ramified microglia in cortical tissue; however, there was great variability in the intensity of staining between individual cells, with some cells possessing very faint to indiscernible staining. In both fields of cells as well as individual cells, OX-42 staining could be observed to co-localize with Lucifer yellow pinocytotic labelling (Fig. 3). The Lucifer yellow labelling displayed the typical feature of sequestration in pinocytotic vesicles, while bright OX-42 fluorescence was associated with the surface membrane. Thus, the pinocytotic cells were confirmed as microglia. Similar resuits were also obtained in some instances using another monoclonal antibody (ED1) specific for macrophages [4, 9]. The ramified microglia in brain tissue have been

demonstrated in the present studies to show a higher level of pinocytotic activity than the surrounding cells. This finding is consistent with other reports indicating pinocytotic activity in these cells [6, 15], and extends the previous studies through the use of whole or intact tissue in conjunction with a specific microglial marker to confirm identification. While microglia were definitely labelled via pinocytosis and the ramified form normally predominates in adult tissue, the possibility that some of the observed cells may be active macrophages derived from ramified microglia cannot be eliminated. This was further suggested by a uniformly round appearance of some cells and occasional staining with ED1, which stains microglia in culture but does not appear to normally stain the ramified form in situ [9]. Thus, some cells may be activated or reactive microglia; however, some of the labelled cells possessed distinctly ramified morphology (see Fig. 2), establishing the presence of pinocytosis in ramified cells prior to activation. The same conclusion was reached by McKenna [15], and this is also in agreement with our previous findings in culture [23]. In conclusion, the observation of pinocytotic activity and motility in ramified microglia in tissue culture pre-

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Fig. 3. Co-localization of pinocytotic activity with OX-42 immunohistochemical staining. A,B: the same field is shown in both, exhibiting Lucifer yellow pinocytosis in A and rhodamine fluorescenceof OX-42 staining in B. Note that most labelled cells contain both types of staining. C,D: an individual cell from the field in A and B (indicated by arrow in each) is shown at higher magnification to depict the features of staining with Lucifer yellow and for OX-42 antigen. Bar =70 pm (A,B) and 15 pm (C,D).

viously led us to propose that these cells m a y f u n c t i o n to sequester soluble c o m p o n e n t s (neuroactive comp o u n d s , metabolic b y p r o d u c t s etc.). Pinocytotis in ramified cells in b r a i n tissue is reported here. This activity c o m b i n e d with the regular d i s t r i b u t i o n o f these cells t h r o u g h o u t the tissue m a y enable them to serve as a filtering system to remove c o m p o u n d s from the extracellular fluid (i.e. fluid cleansing). Assistance from Dr. D.L. Kachele with H R P procedures is appreciated; gratitude is also expressed to Dr. F.L. J o r d a n for s u p p o r t a n d assistance t h r o u g h o u t this work. Supported by the O S U College of Dentistry a n d a K l i n g e n s t e i n Fellowship in the Neuroscience (W.E.T.). 1 Bocchini, V., Artault, J.C., Rebel, G., Dreyfus, H. and Massarelli, R., Phagocytosis of polystyrene latex beads by rat brain microglia cell cultures is increased by treatment with gangliosides,Dev. Neurosci., 10 (1988) 270-276. 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.

3 Colton, C.A. and Gilbert, D.L., Production of superoxide anions by a CNS macrophage, the microglia, FEBS Lett., 223 (1987) 284288. 4 Dijkstra, C.D., Dopp, E.A., Joling, P. and Kraal, G., The heterogeneity of mononuclear phagocytes in lymphoid organs; distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3, Immunology, 54 (1985) 589-599. 5 Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A., Antigen presentation and tumor cytotoxicity by interferon-7-treated microglial cells, Eur. J. Immunol., 17 (1987) 12711278. 6 Gebicke-Haerter, P.J., Bauer, J., Schobert, A. and Northoff, H., Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglialceils,J. Neurosci., 9 (1989) 183-194. 7 Gebicke-Haerter, P.J., Rich, I.N., Schobert, A. and Northoff, H., Regulation of microglial cytokine expression. Int. J. Dev. Neurosci., 8-S1 (1990) 79 (Abstr. 78). 8 Glenn, J.A., Jordan, F.L. and Thomas, W.E., Further studies on the identification of microglia in mixed brain cell cultures, Brain Res. Bull., 22 (1989) 1049-1052. 9 Graeber, M.B., Banati, R.B., Streit, W.J. and Kreutzberg, G.W., Immunophenotypic characterization of rat brain macrophages in cultures, Neurosci. Lett., 103 (1989) 241-246.

31 10 Hayes, G.M.., Woodroofe, M.N. and Cuzner, M.L., Characterization of microglia isolated from adult human and rat brain, J. Neuroimmunol., 19 (1988) 177-189. 11 Hetier, E., Ayala, J., Denefle, P., Bousseau, A., Rouget, P., Mallat, M. and Prochiantz, A., Brain macrophages synthesize interleukin-1 and interleukin-I mRNAs in vitro, J. Neurosci. Res., 21 (1988) 391-397. 12 Hickey, W.F. and Kimura, H., Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo, Science, 239 (1988) 290-292. 13 Jordan, F.L. and Thomas, W.E., Identification of somatostatincontaining neurons in primary cultures of rat cerebral cortex, Neurosci. Lett., 77 (1989) 249-254. 14 Jordan, F.L. and Thomas, W.E., Brain macrophages: questions of origin and interrelationship, Brain Res. Rev., 13 (1988) 165-178. 15 McKenna, O.C., Endocytic activity of subependymal microglial cells in the toad brain: a cytochemical study of peroxidase uptake, J. Comp. Neurol., 187 (1979) 169-190. 16 Perry, V.H. and Gordon, S., Macrophages and microglia in the nervous system, Trends Neurosci., 11 (1988) 273-277. 17 Perry, V.H., Hume, D.A. and Gordon, S., Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain, Neurosience, 15 (1985) 313-326.

18 Rieske, E., Graeber, M.B., Tetzlaff, W., Czlonkowska, A., Streit, W.J. and Kreutzberg, G.W., Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro, Brain Res., 492 (1989) 1-14. 19 Rio-Hortega, P., Microglia. In W. Penfield (Ed.), Cytology and Cellular Pathology of the Nervous System, Vol. 2, Hocker, New York, 1932, pp. 481-584. 20 Robinson, A.P., White, T.M. and Mason, D.W., Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3, Immunology, 57 (1986) 239-247. 21 Sasaki, A., Levison, S.W. and Ting, J.P.-Y., Comparison and quantitation of Ia antigen expression on cultured macroglia and ameboid microglia from Lewis rat cerebral cortex: analyses and implications, J. Neuroimmunol., 25 (1989) 63-74. 22 Suzumura, A., Mezitis, S.G.E., Gonatas, N.K. and Silberberg, D.H., MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by y-interferon, J. Neuroimmunol., 15 (1987) 263-278. 23 Thomas, W.E., Properties of ramified microglia in rat cerebral cortical cell cultures, Int. J. Dev. Neurosci., 8-S1 (1990) 79 (Abstr. 79).

Pinocytotic activity in ramified microglia.

Pinocytotic activity was investigated in rat cerebral cortex using the soluble tracers horseradish peroxidase and Lucifer yellow. A subpopulation of c...
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