Journal of Neuroscience Research 29:13-28 (1991)
Characterization of Ramified Microglia in Tissue Culture: Pinocytosis and Motility S.A. Ward, P.A. Ransom, P.L. Booth, and W.E. Thomas Department of Oral Biology, College of Dentistry, Ohio State University, Columbus, Ohio
Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis? and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: y-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through timelapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.
by Rio-Hortega (1932), who developed a silver carbonate method to stain these cells selectively and used this procedure to study their functional role. From these early studies, Rio-Hortega suggested that the microglia served as macrophages or phagocytic cells. While this hypothesis has persisted for some time, only recently has it received substantial support. Several laboratories indicate that microglial cells are derived from the usual macrophage precursor: blood monocytes (Fujita et al., 1981; Imamoto, 1981; Ling, 1981). Microglia have been shown to be selectively stained by several monoclonal antibodies that specifically label macrophages, including F4/80 (antigen unknown), 2.462 (to Fc receptors), and MAC-1 (to CR3 receptors) (Perry et al., 1985; Perry and Gordon, 1987, 1988). Using rat bone marrow chimeras, Hickey and Kimura (1988) showed that in addition to being derived from bone marrow cells, microglia were also capable of antigen presentation leading to lymphocyte activation. Finally, evidence has been presented that the microglia express phagocytic activity in response to tissue injury (Brierley and Brown, 1982). Thus, much recent work appears to support the initial hypothesis of Rio-Hortega that microglia are intrinsic macrophages of the CNS. At least three forms or types ofmicroglia have been identified in situ: ramified, amoeboid, and reactive cells (reviewed in Jordan and Thomas, 1988). The ramified form, also termined vesting microglia, is the normal constituent of healthy adult brain tissue (Mori and Leblond, 1969; Maurabe and Sano, 1981c, 1982b). Ramified microglia are characterized by a unique morphology, exhibiting a small, usually oval, soma with numerous highly branching thin processes; these processes often display an overall prickly or spiny appearance (Murabe and Sano, 1981a,c; Perry et al., 1985; Perry and Gordon, 1988). The amoeboid form of microglia has a quite dif-
Key words: ramified microglia, rat, cerebral cortex, video microscopy, fluid cleansing INTRODUCTION Microglia were originally established as a distinct cell population within the central nervous system (CNS) 0 1991 Wiley-Liss, Inc.
Received June 12, 1990; revised September 27, 1990; accepted October 3, 1990. Address reprint requests to Dr. W. Eric Thomas, Oral Biology Department, College of Dentistry, Postle Hall, 305 w. 12th Avenue, Ohio State University, Columbus, OH 43210.
14
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ferent appearance, displaying a larger, irregular-shaped, or pleomorphic cell body with pseudopodia, and lacking the branching processes of the ramified form (Ling, 1976a,b; Murabe and Sano, 1982b; Tseng et al., 1983). Amoeboid cells are clearly established as a transient form during the late prenatal to early postnatal period of development (Ivy and Killackey, 1978; Murabe and Sano, 1982b, 1983; Innocenti et al., 1983a,b). The reactive microglia are small round cells devoid of processes (Matthews and Kruger, 1973; Murabe et al., 1981a); they are also a transient population that arises in adult brain in response to tissue injury or damage (Matthews and Kruger, 1973; Murabe et al., 1981a,b, 1982). Reactive microglia have been referred to by several other names, including rod cells and gitter cells (Jordan and Thomas, 1988). These different forms of microglial cells-ramified, amoeboid, and reactive-appear to correspond to varying functional states of a single cell type or class, as the amoeboid cells have been indicated to convert into ramified microglia (Ling, 1976b; Imamoto, 1981; Murabe and Sano, 1982b; Giulian and Baker, 1986) and ramified cells suggested to give rise to reactive microglia (Torvik, 1975; Murabe et al., 1981a, 1982; Brierley and Brown, 1982). In addition, while ramified microglia possess some macrophage markers, these cells do not directly appear to be active macrophages (Brierley and Brown, 1982; Giulian and Baker, 1986; Giulian, 1987; Perry and Gordon, 1987). However, both in vivo and in vitro studies support the inference that amoeboid (Ling, 1976b, 1977; Ling et al., 1983; Giulian and Baker, 1986; Giulian, 1987) and reactive (Matthews and Kruger, 1973; Torvik, 1975; Brierley and Brown, 1982; Frei et al., 1987) microglia are indeed active macrophages. These properties have contributed to the development of the view that ramified microglia are inactive or dormant macrophages (i.e., resting) that serve as precursors for the production of active macrophage cells. In this view, ramified microglia are derived from monocytic blood cells during development with the amoeboid form as an active macrophage intermediate; they are converted into reactive cells when active macrophages are needed in adult tissue in response to injury. This overall scheme is consistent with the bulk of the existing information and explains several factors, including the relationship of the various microglial forms, the source of macrophages in CNS tissue, and the functional role of amoeboid and reactive cells; however, it appears to leave the ramified microglia without a direct function. While they may serve as precursors for active macrophages, in the absence of a requirement for such cells, ramified microglia are generally considered to be quiescent. Thus, for the form of microglia normally present in adult tissue and that comprises a significant
portion of the cell population, no constitutive function has been clearly established. Our laboratory recently successfully identified ramified microglia in tissue cultures of rat cerebral cortical cells (Glenn et al., 1989). This preparation appears to provide a good opportunity to investigate intrinsic functional properties specifically of the ramified form of microglia. In the initial studies reported here, endocytotic activity was investigated in these cells. While ramified microglia are thought not to possess phagocytic capability (Torvik, 1975; Brierlley and Brown, 1982; Giulian, 1987), this is mainly based on studies in vivo in which this activity can be difficullt to determine. Thus, it seems possible that ramified cells; may have residual or a lower level of phagocytic activity. In addition, amoeboid microglia appear to exhibit significant pinocytotic activity as they label by ingesting soluble compounds, particularly horseradish peroxidase (HIIP) (Ivy and Killackey , 1978; Valentino and Jones, 1981; Innocenti et al., 1983b; Leong et al., 1983). Both phagocytosis and pinocytotic activity were assessed in the present work; the results suggest one potential constitutive function of ramified microglia. A portion of this work has been presented in a preliminary form (Glenn et al., 1990).
MATERIALS AND METHODS Tissue Culture The preparation and maintenance of primary cultures of cerebral cortical cells have been previously described (Thomas, 1985; Jordan let al., 1987); however, an overview of the methods employed is provided here. Cerebral cortical tissue was removed from fetal animals taken directly from the uterus of time-pregnant rats (Sprague-Dawley) during gestational days 14-17. Tissue was dissected from both cerebral hemispheres, rinsed extensively in modified culture medium, and the pooled pieces dissociated by trituration 'in calcium- and magnesium-free Hanks' balanced salt solution (HBSS). The resulting cell suspension in the supernatant fraction was removed and a cell count performed. Cells were plated at an approximate density of 5 x lo4 cells/cm* on a collagenipolylysine-coated surface nn modified 35-mm tissue culture dishes. The growth medium for cultures consisted of Eagle's minimal essential medium (MEM) supplemented with 0.7% Methcicel, 5% rat serum, 17 mM glucose, 2 mM glutamine,
Characterization of Ramified Microglia in Tissue Culture: Pinocytosis and Motility S.A. Ward, P.A. Ransom, P.L. Booth, and W.E. Thomas Department of Oral Biology, College of Dentistry, Ohio State University, Columbus, Ohio
Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis? and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: y-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through timelapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.
by Rio-Hortega (1932), who developed a silver carbonate method to stain these cells selectively and used this procedure to study their functional role. From these early studies, Rio-Hortega suggested that the microglia served as macrophages or phagocytic cells. While this hypothesis has persisted for some time, only recently has it received substantial support. Several laboratories indicate that microglial cells are derived from the usual macrophage precursor: blood monocytes (Fujita et al., 1981; Imamoto, 1981; Ling, 1981). Microglia have been shown to be selectively stained by several monoclonal antibodies that specifically label macrophages, including F4/80 (antigen unknown), 2.462 (to Fc receptors), and MAC-1 (to CR3 receptors) (Perry et al., 1985; Perry and Gordon, 1987, 1988). Using rat bone marrow chimeras, Hickey and Kimura (1988) showed that in addition to being derived from bone marrow cells, microglia were also capable of antigen presentation leading to lymphocyte activation. Finally, evidence has been presented that the microglia express phagocytic activity in response to tissue injury (Brierley and Brown, 1982). Thus, much recent work appears to support the initial hypothesis of Rio-Hortega that microglia are intrinsic macrophages of the CNS. At least three forms or types ofmicroglia have been identified in situ: ramified, amoeboid, and reactive cells (reviewed in Jordan and Thomas, 1988). The ramified form, also termined vesting microglia, is the normal constituent of healthy adult brain tissue (Mori and Leblond, 1969; Maurabe and Sano, 1981c, 1982b). Ramified microglia are characterized by a unique morphology, exhibiting a small, usually oval, soma with numerous highly branching thin processes; these processes often display an overall prickly or spiny appearance (Murabe and Sano, 1981a,c; Perry et al., 1985; Perry and Gordon, 1988). The amoeboid form of microglia has a quite dif-
Key words: ramified microglia, rat, cerebral cortex, video microscopy, fluid cleansing INTRODUCTION Microglia were originally established as a distinct cell population within the central nervous system (CNS) 0 1991 Wiley-Liss, Inc.
Received June 12, 1990; revised September 27, 1990; accepted October 3, 1990. Address reprint requests to Dr. W. Eric Thomas, Oral Biology Department, College of Dentistry, Postle Hall, 305 w. 12th Avenue, Ohio State University, Columbus, OH 43210.
14
Ward et al.
ferent appearance, displaying a larger, irregular-shaped, or pleomorphic cell body with pseudopodia, and lacking the branching processes of the ramified form (Ling, 1976a,b; Murabe and Sano, 1982b; Tseng et al., 1983). Amoeboid cells are clearly established as a transient form during the late prenatal to early postnatal period of development (Ivy and Killackey, 1978; Murabe and Sano, 1982b, 1983; Innocenti et al., 1983a,b). The reactive microglia are small round cells devoid of processes (Matthews and Kruger, 1973; Murabe et al., 1981a); they are also a transient population that arises in adult brain in response to tissue injury or damage (Matthews and Kruger, 1973; Murabe et al., 1981a,b, 1982). Reactive microglia have been referred to by several other names, including rod cells and gitter cells (Jordan and Thomas, 1988). These different forms of microglial cells-ramified, amoeboid, and reactive-appear to correspond to varying functional states of a single cell type or class, as the amoeboid cells have been indicated to convert into ramified microglia (Ling, 1976b; Imamoto, 1981; Murabe and Sano, 1982b; Giulian and Baker, 1986) and ramified cells suggested to give rise to reactive microglia (Torvik, 1975; Murabe et al., 1981a, 1982; Brierley and Brown, 1982). In addition, while ramified microglia possess some macrophage markers, these cells do not directly appear to be active macrophages (Brierley and Brown, 1982; Giulian and Baker, 1986; Giulian, 1987; Perry and Gordon, 1987). However, both in vivo and in vitro studies support the inference that amoeboid (Ling, 1976b, 1977; Ling et al., 1983; Giulian and Baker, 1986; Giulian, 1987) and reactive (Matthews and Kruger, 1973; Torvik, 1975; Brierley and Brown, 1982; Frei et al., 1987) microglia are indeed active macrophages. These properties have contributed to the development of the view that ramified microglia are inactive or dormant macrophages (i.e., resting) that serve as precursors for the production of active macrophage cells. In this view, ramified microglia are derived from monocytic blood cells during development with the amoeboid form as an active macrophage intermediate; they are converted into reactive cells when active macrophages are needed in adult tissue in response to injury. This overall scheme is consistent with the bulk of the existing information and explains several factors, including the relationship of the various microglial forms, the source of macrophages in CNS tissue, and the functional role of amoeboid and reactive cells; however, it appears to leave the ramified microglia without a direct function. While they may serve as precursors for active macrophages, in the absence of a requirement for such cells, ramified microglia are generally considered to be quiescent. Thus, for the form of microglia normally present in adult tissue and that comprises a significant
portion of the cell population, no constitutive function has been clearly established. Our laboratory recently successfully identified ramified microglia in tissue cultures of rat cerebral cortical cells (Glenn et al., 1989). This preparation appears to provide a good opportunity to investigate intrinsic functional properties specifically of the ramified form of microglia. In the initial studies reported here, endocytotic activity was investigated in these cells. While ramified microglia are thought not to possess phagocytic capability (Torvik, 1975; Brierlley and Brown, 1982; Giulian, 1987), this is mainly based on studies in vivo in which this activity can be difficullt to determine. Thus, it seems possible that ramified cells; may have residual or a lower level of phagocytic activity. In addition, amoeboid microglia appear to exhibit significant pinocytotic activity as they label by ingesting soluble compounds, particularly horseradish peroxidase (HIIP) (Ivy and Killackey , 1978; Valentino and Jones, 1981; Innocenti et al., 1983b; Leong et al., 1983). Both phagocytosis and pinocytotic activity were assessed in the present work; the results suggest one potential constitutive function of ramified microglia. A portion of this work has been presented in a preliminary form (Glenn et al., 1990).
MATERIALS AND METHODS Tissue Culture The preparation and maintenance of primary cultures of cerebral cortical cells have been previously described (Thomas, 1985; Jordan let al., 1987); however, an overview of the methods employed is provided here. Cerebral cortical tissue was removed from fetal animals taken directly from the uterus of time-pregnant rats (Sprague-Dawley) during gestational days 14-17. Tissue was dissected from both cerebral hemispheres, rinsed extensively in modified culture medium, and the pooled pieces dissociated by trituration 'in calcium- and magnesium-free Hanks' balanced salt solution (HBSS). The resulting cell suspension in the supernatant fraction was removed and a cell count performed. Cells were plated at an approximate density of 5 x lo4 cells/cm* on a collagenipolylysine-coated surface nn modified 35-mm tissue culture dishes. The growth medium for cultures consisted of Eagle's minimal essential medium (MEM) supplemented with 0.7% Methcicel, 5% rat serum, 17 mM glucose, 2 mM glutamine,
