Retinoic acid enhances the secretion of plasminogen from cultured rat microglia

Abstract

To determine the amount of plasmmogen in microghal condmoned medium, a highly sensitive and Sl~Clfic enzyme-linked immuno~orbent assay (ELISA) for lot plasmmogen wa~ e~tablished. Weak cross-reactwity with human serum plasminogen was observed, while no re.activity was deified with frog and carp plasmmogen. The specificity of the immuno~orbent a~say was confirmed by Western blotting. The secretion of plasmmogen into the microglial culture medium was ouantlfied by using the established ELISA and was found to be increased depending on the culture time and numbe~ of microgha The secretion was increased about 5-fold by stimulation with retmoic acid. while interleukm-1, and basic fibroblast growth factor showed no significant effect.Microgha; Plasmmogen, Enzyme-li!~ed immunosorbent assay, Culture; Conditioned medium 1, INTRODUCTIONMicroglia have been thought to be implicated not only in regeneration after injury or pathological damage but also in morphogenesis during development of the central nervous system (CNS) [1]. To study these events, methods for isolation of microglia and an in vitro culture system have been developed [2][3][4][5]. So far, isolated and cultured microglia have been reported to produce the biologically active substances: nerve growth factor (1NGF) [6], interleukin-1 (1L-I) [7]~ IL-6 [8], tumor necrosis factor (TNF) [9,10] and basic fibroblast growth factor (bFGF) [11]. Furthermore, we recently found that microglia secrete some serine-type pretenses, which were identified as elasta~e [12] and a urokinase-type plasminogen activator fuPA) [13]. More recently, plasminogen, which is a specific substrata of PA0 was also detected in microglial conditioned medium (Mic-CM) by zymography and Western blotting [14]. The de nero synthesis of plasminogen was also demonstrated in microglia [14]. However, how plasminogen secretion from microglia is regulated remained unknown. To obtain further information about microglia-derived plasminogen, we investigated the eflbct of retinoic acid° which is known to accelerate microglial differentiation [2], on the secretion of plasminogen from microglia. The effect of bFGF and iL-1, as stimulators of PA secretion by microglia [13], was also investigated. MATERIALS AND METHODS 1. Preparr.tton of mtc,oghtt! conditioned nledfumThe isolation of rat mieroglia from a primary culture of Day 1 rat brain and the preparation of mieroghal conditioned medium (Mic-CM) were described previously [5,12,13]. Mleroglial galls were allow~l to adhere to 6-or 12-well plates (Costar). After removal of nonattached ~ils, the miereglia were washed 3 times with serum-free Dulbeoeo'~ modified Eagle medium (DMEM), and incubated with 0 5 ml (for 12-well plates) or 1 ml (for 6-well plates) of the ~ame medium in the presence or absence of retinoie acid, IL-I (Genzymg)and bFGF (R&D Systems). The recovered conditioned medmm was centrifuged at 1,500 rpm rot 10 mm and tim supernatant was used for ELISA either ~mmediately or after storage at -80"C. Pur~icat/on of plazntttwgen attd preparation of the antibody...

Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro

“…7). We investigated the antiapoptotic activities of 1 ng/ml of IL-3, IL-6, M-CSF, bFGF, and TNF␣ and 10 µg/ml of PGn, which are produced by cultured microglial cells (Shimojo et al, 1991;Nakajima et al, 1992;Lee et al, 1993;Appel et al, 1995;Sawada et al, 1995). Figure 7 shows that PGn and TNF␣ were effective in rescuing neurons from SNP-induced neuronal apoptosis, whereas the other cytokines were ineffective.…”

“…PGn has been a candidate for neurotrophic factors secreted by microglial cells (Nagata et al, 1993), and it was revealed to have neuroprotective activity against NO-induced injury in the present study, but a rather high concentration of PGn (10 µg/ml) was necessary to preclude neuronal apoptosis. According to the study of Nakajima et al (1992), MiCM contains PGn at concentrations less than 1 ng/ml. Moreover, the neuroprotective action of PGn was abolished completely by heat treatment.…”

Multifunctional roles of enolase in Alzheimer’s disease brain: beyond altered glucose metabolism

“…2004a), and consequently, propagating an overly sensitive inflammatory response. Alternatively, activated microglia and apoptotic neuronal cells also synthesize PGn, in addition to tPA (Nakajima et al. 1992a,b,c; Tsirka et al.…”

Neurotrophins regulate the function of cultured microglia

“…ED-1 monoclonal antibody was obtained from Serotec (Oxford, UK). The antibody against rat plasminogen was raised in rabbits and further purified as IgG by using ImmunoPure (Pierce, Rockford, IL) (Nakajima et al, 1992b). Anti-rat/ mouse NGF receptor monoclonal antibody was purchased from Boehringer Mannheim (Germany).…”