Mitogenic effects of interleukin-5 on microglia

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NeuroscienceLetters201 (1995)131-134

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Mitogenic effects of interleukin-5 on microglia Garth E. Ringheim* Neuroscience Therapeutic Domain, HoechstoRoussel Pharmaceuticals, Inc., Route 202-206 N, Room L-219, Somerville, NJ 08876, USA

Received22 September1995; revisedversionreceived23 October1995; accepted23 October1995

Abstract Interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte macrophage-colony stimulating factor (GM-CSF) are cytokines that bind to receptor complexes comprised of unique a-receptor subunits specific for each ligand and a commonly shared t-receptor subunit. Previous studies have shown that IL-3 and GM-CSF induce mitosis in microglia and macrophage cells, indicating the functional presence of their cognate receptors. In this study, it is shown that the third member of this cytokine group, IL-5, also serves as a microglia mitogen. Proliferative effects were seen in culture on both murine microglia and a murine macrophage cell line, RAW 264.7. Since IL-5 is known to be secreted by both microglia and astrocytes in response to inflammatory stimuli, these results indicate that IL-5 may be involved in the cytokine-immune cascades leading to microglia proliferation in areas affected by disease and tissue damage. Keywords: Interleukin-5; Microglia; Mouse; Macrophage

Microglia are macrophage-like cells in the brain capable of migrating to various regional locations, where they take on roles as diverse as debris removal, pathogen defense, cell destruction, and neuronal support (for review, see [5,14]). In carrying out these functions, this cell type displays a variety of dynamic activities, one of which is to proliferate in response to signals from the extracellular environment. Under normal conditions, only low basal levels of proliferation have been described for microglia in vivo [12]. In contrast, extensive microglia proliferation has been observed at certain developmental stages of brain formation [25] and from immunological activation [13,16]. In culture, more defined stimulus conditions have been used to elucidate the factors regulating microglia proliferation. Inhibiting effects have been described for lipopolysaccharide (LPS) [9] and transforming growth factorbeta (TGF-fl) [22], whereas mitogenic effects have been described for microglia treated with beta amyloid [2], colony stimulating factor-1 [1,20], interleukin-3 (IL-3), granulocyte macrophage-colony stimulating factor (GMCSF) [8-10] and, at least in mixed glial cultures, interleukin- lfl (IL- lfl) [9].

* Tel.: +] 908 2314928; fax: +1 908 2314335; e-mail: [email protected].

Since interleukin-5 (IL-5) is known to share a common t-receptor component with the receptors that bind the known microglia mitogens IL-3 and GM-CSF [6,23], IL5 was hypothesized in this study to also have mitogenic effects on microglia and macrophages. To test this hypothesis, cultured murine microglia and RAW 264.7 murine macrophage cells (American Type Culture Collection, Rockville, MD, USA) were treated with IL-3, IL5 and GM-CSF as well as with LPS, IL-lfl, IL-2, IL-6 and TNF-a, and their proliferative responses measured and compared. Microglia were prepared from pooled cortices of 35 day old newborn mice (strain ND4; Harlan SpragueDawley, Inc., Indianapolis, IN, USA). Briefly, the meninges were dissected away from the cortex and the tissue mechanically triturated in 10 ml Hank's Buffered Saline Solution without calcium or magnesium (HBSS; GIBCO Life Technologies Inc., Grand Island, NY, USA). The cells were washed once in 50 ml HBSS and seeded at 5 x 106 viable cells in poly-L-lysine (Sigma, St. Louis, MO, USA) coated T-150 cm 2 flasks containing 20 ml of culture medium (DMEM/F12 plus 10% fetal calf serum, 50 U/ml each of penicillin and streptomycin, 4 mM glutamine, 1 mM sodium pyruvate, and 15 mM HEPES; GIBCO Life Technologies Inc., Grand Island, NY, USA). After allowing cells to adhere 48 h without distur-

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bance, the medium was removed, the cells rinsed once with phosphate buffered saline, and 20 ml of fresh culture medium added. On day 5, the culture medium was replaced with 20 ml fresh medium and the flask left undisturbed until use. On culturing day 14, microglia were shaken off the astrocyte layer and seeded at 1 x 104 cells in 100btl culture medium in uncoated 96-well plates, and grown overnight. Purity of the cultures was assessed by immunostaining with antibodies to astrocyte GFAP and microglial mac-1 antigens (Boehringer-Mannheim, Indianapolis, IN, USA) and to the F4/80 microglial antigen (BISOURCE International, Camarillo, CA, USA) and judged to be 95-98% pure. The day following plating into 96-well dishes, medium was removed and the cells grown for 48 h with 100/A of fresh medium in the absence or presence of lipopolysaccharide (LPS; Escherichia coli strain 055:B5; Sigma, St. Louis, MO, USA) or the indicated concentrations of the murine cytokines IL-lfl, IL-2, IL-3, IL-5, IL-6, TNF-a, or GM-CSF. To inhibit IL-5-induced microglia proliferation or to test possible autocrine production of IL-3, IL-5, or GM-CSF by microglia, goat-anti-murine polyclonal antibodies to IL-3, IL-5, or GM-CSF (R&D Systems, Minneapolis, MN, USA) were added to the culture medium at 10/zg/ml for 1 h prior to dosing cells. All cytokines used except IL-5 were from BIOSOURCE International (Camarillo, CA, USA). IL-5 was from R&D Systems (Minneapolis, MN, USA). During the last 24 h of incubation, 25/zl of culture medium containing 0.5/zCi of tritiated thymidine (20 Ci/mmol; DuPont NEN, Boston, MA, USA) was added to each well. After a total incubation of 48 h, the medium was removed and the cells lysed with 100 mM NaOH, filtered with water onto 96-well GF/C filter plates (Packard, Meriden, CT, USA), and counted on a Packard Top Count scintillation counter. RAW 264.7

cells were grown in the same medium and stimulation conditions as described for the microglia, except that proliferation was measured using the MTS mitochondrial activity assay at the 48 h incubation time as described by the manufacturer of the MTS kit (Promega, Madison, WI, USA). Proliferation values were compared using the Student's t-test and were considered significant at P < 0.05. Since these cells all incorporated bromodeoxyuridine as viewed immunohistochemically (data not shown), more quantitative methods using thymidine incorporation and MTS activity were used to measure microglia and RAW 264.7 proliferation responses, respectively. For microglia, a basal level of proliferation of microglia was observed to occur under the conditions used in these experiments with basal levels of both thymidine (4000 cpm) and bromodeoxyuridine incorporation being observed (data not shown). IL-lfl, IL-2 and TNF-a had no effect on measured basal activity, whereas IL-6 inhibited slightly and LPS completely abrogated mitotic activity (Fig. 1). In agreement with our results, Ganter et al. [9] reported no mitogenic effects for either IL-lfl or TNF-a on purified microglial cultures, although they did observe microglia proliferation in mixed glial cultures, probably as a result of other factors secreted by the astrocytes. As expected, a dose-response relationship was observed for IL-3 and GM-CSF on both microglia and RAW 264.7 proliferation

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Medium LPS IL-I[~ IL-2 IL-6 TNF-cc Fig. 1. Effects of LPS, IL-1/~, IL-2, IL-6, and TNF-a on the proliferative potential of murine microglia. The concentrations used for stimulation of microglia with LPS and cytokines were based on literature reports of biological activity for each respective stimulus. Cells were incubated 48 h with IL-I~ (5 nM), IL-2 (1 nM), IL-6 (1 nM), and TNFct (1 nM) and thymidine incorporation measured over the last 24 h. Data are represented as the mean -+ SEM of six replicates. *P < 0.05 as compared to control medium.

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Cytokine (mol/L) Fig. 2. Proliferative effects of IL-3, IL-5, and GM-CSF on microglia and RAW 264.7 macrophage cells. (A) Microglia incorporation of [3H]thymidine added in the last 24 h of a 48 h incubation of the cells with the indicated concentrations of IL-3 (o), IL-5 (O), and GM-CSF (El). (B) RAW 264.7 murine macrophage proliferation as measured by the mts assay after a 48 h incubation with the indicated concentrations of IL-3 (o), IL-5 (O), and GM-CSF (1"1). Data are represented as the mean _+SEM of six replicates.

G.E. Ringheim / Neuroscience Letters 201 (1995) 131-134

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Fig. 3. Antibody inhibition of basal and IL-5-stimulated levels of microglia proliferation. Microglia were grown in the absence (lane 1) or presence of 100 pM IL-5 (lane 2), 100 pM IL-5 plus 10/~g/ml anti-IL-5 (lane 3), 10/~g/ml anti-IL-5 (lane 4), 10/~g/ml anti-lL-3 (lane 5), or 10/~g/ml anti-GM-CSF (lane 6) for 48 h. Data are represented as the mean _+ SEM of six replicates. *P < 0.05 as compared to control medium.

(Fig. 2). Maximal levels of stimulation approached 50% for both cell types. Similarly, IL-5 also induced nearly a 50% increase in proliferation with both cell types except that the dose-response curve was more gradual in the case of microglia. To our knowledge, this is the first report demonstrating mitogenic activity for IL-5 on microglia or other macrophage-like cells. The mitogenic effects of IL-3 and GM-CSF have been previously described [8-10]. The receptors for IL-3 and GM-CSF belong to a large family of hematopoietic receptors that includes the IL-5 receptor [4,7]. The receptor complexes for IL-3, IL-5, and GM-CSF all have separate a-receptor subunits to which they bind, but they share a common fl-receptor subunit essential for the formation of functional high affinity receptor complexes [6,11,23]. As seen by the data presented here, both microglia and RAW 264.7 macrophage cells respond to all three of these cytokines, suggesting the presence of the specific a-receptor subunits as well as the shared fl-receptor subunit. Several physiological sources for IL-5 have been described. IL-5 is a cytokine secreted by T-cells [24] and mast cells [15], and is known to play a role in the activation and proliferation of both eosinophils [17,21] and Bcells [18]. More recently, IL-5 has been identified as being a factor secreted by primary mouse astrocytes and microglia in culture [1911. The low level of basal IL-5 secretion from microglia in culture is consistent with the low levels of thymidine and bromodeoxyuridine incorporation that we observed in our study. In this sense, IL-5 would be acting in an autocrine regulatory fashion. In fact, adding antibodies to IL-5 inhibited the level of basal proliferation as well as the IL-5-stimulated proliferation observed in the microglia cultures by 65% and 70%, respectively (Fig. 3, lanes 3 and 4). IL-3 antibodies had no effect (Fig. 3, lane 5) and GM-CSF antibodies inhibited thymidine incorporation by 35% (Fig. 3, lane 6).

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Few roles have been ascribed to IL-5 in the brain. One study has shown that IL-5 induces the secretion of nerve growth factor from astrocytes in culture [3], implicating an indirect involvement in neurotrophic activities in the brain. In the study presented here, another role for IL-5 in the central nervous system could be as a mitogenic factor supporting microglia growth. Since immune activating cytokines such as IFN-y induce astrocyte secretion of IL5 [19], it is conceivable that some of the proliferative responses of microglia observed in vivo after injury or infection are from the mitogenic activity of IL-5, as well as other known stimulators of proliferation such as IL-3 and GM-CSF. [1] Alliot, F., Lecain, E. Grima, B. and Pessac, B., Microglial progenitors with a high proliferative potential in the embryonic and adult mouse brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 15411545. [2] Aranjo, D.M. and Cotman, C.W., Beta-amyloid stimulates glial cell in vitro to produce growth factors that accumulate in senile plaques in Alzheimer's disease, Brain Res., 569 (1992) 141-145. [3] Awatsuji, H., Furukawa, Y., Hirota, M., Murakami, Y., Nii, S., Furukawa, S. and Hayashi, K., lnterleukin-4 and -5 as modulators of nerve growth factor synthesis/secretion in astrocytes, J. Neurosci. Res., 34 (1993) 539-545. [4] Bazan, J.F., Structural design and molecular evolution of a cytokine receptor family, Proc. Natl. Acad. Sci. USA, 87 (1990) 6934-6938. [5] Davis, E.J., Foster, T.D. and Thoma, W.E., Cellular forms and functions of brain microglia, Brain Res. Bull., 34 (1994) 73-78. [6] Devos, R., Plaetinck, G., Van der Heyden, J., Cornelis, S., Vadekerckhove, J., Fiers, W. and Tavernier, J., Molecular basis of a high affinity murine intedeukin-5 receptor, Eur. Mol. Biol. Organ. J., 10 (1991) 2133-2137. [7] Foxwell, M.J., Barrett, K. and Feldmann, M., Cytokine receptors: structure and signal transduction, Clin. Exp. Immunol., 90 (1992) 161-169. [8] Frei, K., Bodmer, S., Schwerdel, C. and Fontana, A., Astrocytederived interleukin 3 as a growth factor for microglial cells and peritoneal macrophages, J. Immunol., 137 (1986) 3521-3527. [9] Ganter, S., Northoff, H., M~innel, D. and Gebicke-H~rter, P.J., Growth control of cultured microglia, J. Neurosci. Res., 33 (1992) 218-230. [10] Giulian, D. and Ingeman, J.E., Colony-stimulating factors as promoters of ameboid microglia, J. Neurosci., 8 (1988) 47074717. [11] Kitamura, T., Sato, N., Arai, K. and Miyajima, A., Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors, Cell, 66 (1991) 1165-1174. [12] Lawson, L.J., Perry, V.H. and Gordon, S., Turnover of resident microglia in the normal adult mouse brain, Neuroscience, 48 (1992) 405-415. [13] Matsumoto, Y., Ohmori, K. and Fujiwara, M., Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system, J. Neuroimmunol., 37 (i 992) 23-33. [14] Perry, V.H., Lawson, L.J. and Reid, D.M., Biology of the mononuclear phagocyte system of the central nervous system and HIV infection, J. Leukoc. Biol., 56 (1994) 399-406. [15] Plaut, M., Pierce, J.H., Watson, J.C., Hanley-Hyde, J., Nordan, R.P. and Paul, W.E., Mast cell lines produce lymphokines in response to cross-linkage of FceRl or to calcium ionophores, Nature, 339 (1989) 64-457.

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