Insulin-like growth factor I and II expression and modulation in amoeboid microglial cells by lipopolysaccharide and retinoic acid

Neuroscience 138 (2006) 1233–1244

INSULIN-LIKE GROWTH FACTOR I AND II EXPRESSION AND MODULATION IN AMOEBOID MICROGLIAL CELLS BY LIPOPOLYSACCHARIDE AND RETINOIC ACID C. KAUR,* V. SIVAKUMAR, S. T. DHEEN AND E. A. LING

entiation (Guan et al., 2003) in the developing nervous system and is most highly expressed by maturing neurons (Bondy and Cheng, 2004). It promotes neuronal survival and has been shown to prevent neuronal apoptosis in vivo and in vitro (Yin et al., 1994; Delaney et al., 2001). IGF-I is also known to be important for glial phenotypic differentiation, oligodendrocyte survival and myelination (Guan et al., 2001; Ye et al., 2002). The role of IGF-II during postnatal development of brain is less clear. It has been proposed that IGF-II could act as a growth and differentiation factor in the CNS since targeted disruption of the IGF-II gene in mice leads to a deficiency in their growth (Kiess et al., 1994). To date, the expression of IGF-I has been described to be predominantly in neurons in the developing nervous system. Recent studies have also reported the expression of IGF-I in activated microglia/macrophages within the damaged areas (O’Donnell et al., 2002; Hwang et al., 2004) during injuries to the CNS. IGF-II is also known to be induced in the developing brain during wound repair following hypoxic ischemic injury (Beilharz et al., 1995). Although immunohistochemical studies have reported the expression of IGF-II in the choroid plexus, cerebral vasculature, ependymal cells, retina, and pituitary gland in the developing brain, its cellular localization remains to be resolved. IGF-I has been considered as essential for brain development during the first 21 postnatal days (Lee et al., 1996) which is the critical period for brain growth in the rat. Besides affecting neuronal maturation and survival, it has been considered as an important factor for oligodendrocyte survival and myelination (Dubois-Dalcq and Murray, 2000; Guan et al., 2001). Although the exact function of IGF-II in the developing brain is not clear, its pattern of association with oligodendrocytes and myelin suggests that it may also play a role in myelination (Logan et al., 1994; Walter et al., 1999). A study which examined myelination during postnatal development in IGF-I knock-out mice found that myelination was reduced by mechanisms which led to a reduction in oligodendrocyte survival and maturation (Ye et al., 2002). These authors also proposed that IGF-II could compensate partly for the actions of IGF-I on myelination. Despite these observations, there is no available evidence on the expression of IGF-I and IGF-II and their cellular localization in the developing brain white matter. In view of this, we sought to determine the gene and protein expression of IGF-I and IGF-II in the developing corpus callosum. We further analyzed the IGF-I and IGF-II expression in amoeboid microglial cells (AMC), con-

Department of Anatomy, Yong Loo Lin School of Medicine, Blk MD10, 4 Medical Drive, National University of Singapore, Singapore 117597

Abstract—Insulin-like growth factors I and II are known to regulate the development of the CNS. We examined the developmental changes in insulin-like growth factor I and insulin-like growth factor II expression in the postnatal rat corpus callosum. Insulin-like growth factor I and insulin-like growth factor II mRNA expression increased at 3 days as compared with 1 day whereas the protein expression increased up to 7 days. Insulin-like growth factor I and insulin-like growth factor II immunoexpression was specifically localized in round cells confirmed by double immunofluorescence with OX-42 to be the amoeboid microglial cells. Insulin-like growth factor I expression was observed up to 7 days in amoeboid microglial cells while insulin-like growth factor II expression was detected in 1–3 day old rats. Exposure of primary rat microglial cell cultures to lipopolysaccharide increased insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression significantly along with their immunoexpression in microglial cells. The lipopolysaccharide-induced increase in insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression was significantly decreased with all-trans-retinoic acid. We conclude that insulinlike growth factor I and insulin-like growth factor II expression in amoeboid microglial cells in the developing brain is related to their activation. Once the activation is inhibited, either by transformation of the amoeboid microglial cells into ramified microglia regarded as resting cells or as shown by the effect of all-trans-retinoic acid administration, insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression is downregulated. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: corpus callosum, microglial cells, insulin-like growth factor I and II, lipopolysaccharide, all-trans-retinoic acid.

Insulin-like growth factors I and II (IGF-I and IGF-II) are peptides which share 50% homology with insulin (Berger, 2001) and are known to regulate the development of the nervous system (Fushimi and Shirabe, 2004). IGF-I plays an important role in promoting cell proliferation and differ*Corresponding author. Tel: ⫹65-65163209; fax: 65-67787643. E-mail address: [email protected] (C. Kaur). Abbreviations: AMC, amoeboid microglial cells; CR3, complement type 3 receptors; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; IGF-I and IGF-II, insulin-like growth factor I and II; IGFBP, insulin-like growth factor binding protein; IGF-IR, insulin-like growth factor-I receptor; LPS, lipopolysaccharide; PBS, phosphatebuffered saline; RA, all-trans-retinoic acid; RT, reverse transcription; TNF-␣, tumor-necrosis factor-␣.

0306-4522/06$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.12.025

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sidered to be active macrophages that are known to preponderate the developing corpus callosum (Kaur et al., 2001). In this connection, it is relevant to note that IGF-I is expressed in activated microglia/macrophages in the adult brain (O’Donnell et al., 2002; Hwang et al., 2004). Since IGF-I and IGF-II expression has been localized in activated microglial cells in brain injuries and, furthermore, IGF-I plays an important role in controlling macrophage cell biology (Renier et al., 1996) it is important to investigate the expression of these growth factors in the AMC. Further, we aimed to examine whether activation or inhibition of AMC activity would alter the mRNA and protein expression of IGF-I and IGF-II in them. This is important as activated macrophages have been reported to release IGF-I providing an extracellular growth and differentiation signal at sites of inflammation (Nagaoka et al., 1990). We also sought to examine the expression of insulin-like growth factor-I receptor (IGF-IR) in the AMC as the actions of IGF-I and IGF-II are reported to be mediated through activation of specific cell surface receptors primarily the IGF-IR (LeRoith et al., 1992). Besides their role as active phagocytes, the presence of microglia/macrophages has been associated with active myelination in the developing brain (Hutchins et al., 1992). In vitro studies have shown that microglia have the ability to induce myelin gene expression in oligodendroglial cultures (Hamilton and Rome, 1994) and macrophage-derived growth factors may play a role in myelinogenesis and myelin repair in inflammatory demyelinating disease (Loughlin et al., 1997). It has been suggested that microglia-macrophages may promote remyelination during a pathological insult in the CNS through the induction of IGF-I (Mason et al., 2001). Lipopolysaccharide (LPS), an outer membrane component of Gram negative bacteria, is a potent activator of macrophages (Meng and Lowell, 1997) and is often used as an agent to study the inflammatory response induced by infections in the CNS (Hagberg and Mallard, 2005). During inflammation, microglia are activated to release cytokines, oxygen free radicals and trophic factors, which will influence the CNS in various ways (Hagberg and Mallard, 2005). Other than this, experimental studies have also shown that the developing brain white matter is extremely sensitive to LPS exposure (Duncan et al., 2002; Mallard et al., 2003). Production of IGF-I by inflammatory macrophages at other sites has been thought to contribute to wound healing (Arkins et al., 1995). Since AMC are macrophages in the developing CNS, we aimed to determine whether IGF-I and IGF-II were expressed by these cells, and if so, whether the expression might be affected when challenged with LPS. Along with this we also aimed to investigate whether the upregulated expression of IGF-I and IGF-II, if any, in AMC activated by LPS would be suppressed by using an agent having an anti-inflammatory effect. We used alltrans-retinoic acid (RA) to suppress AMC activation as it has been reported to have inhibitory effects on microglial activation (Dheen et al., 2005).

EXPERIMENTAL PROCEDURES Animals Fifty-six postnatal 1–21 day old Wistar rats were used in the present study. In the handling and care of animals, the International Guiding Principles for Research as stipulated by WHO Chronicle 39(2):51–56 (1985) and as adopted by the Laboratory Animals Centre, Animal Holding Unit, National University of Singapore were followed. Every effort was made to minimize the number of animals used and their suffering.

Microglial cell culture Mixed glial cultures were prepared from corpus callosum of 1–2 day old postnatal rat brain according to the method of Giulian and Baker (1986) and as described before by Dheen et al. (2005). In brief, cells dissociated from the corpus callosum of rat brain were seeded on a 75-cm2 flask at a density of 1.2⫻106 cells/ml of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C in humidified 5% CO2/95% air. Culture medium was replaced after 24 h and then twice a week. After 2 weeks, microglia were isolated by mild trypsinization and the detached cells were transferred into falcon tubes. After centrifugation (100⫻g) for 5 min, the cells were resuspended in a fresh DMEM supplemented with 10% FBS, plated at a final density of 2.5⫻105 cells/well on a 24 multi-well culture plate and incubated at 37 °C in humidified 5% CO2/95% air for 24 h. After that, the cells were subjected to different treatments. The percentage of microglial cells in the original mixed cultures was about 20% and subsequently 96% in microglial cell cultures established after two weeks. To study the effects of LPS on microglial activation and IGF-I and IGF-II expression, cells were incubated with LPS (1 ␮g/ml) for 6 h. In addition, microglial cells were exposed to RA (10 ␮M, Sigma-Aldrich, MO, USA; Cat. No. R2625) in the presence of LPS for 6 h to analyze the modulation of IGF-I and IGF-II expression in activated microglial cells by RA. The concentration of LPS and RA used was as described by Dheen et al. (2005).

Real time RT-PCR The following materials were used for real time RT-PCR analysis: (1) supraventricular corpus callosum from brains of Wistar rats at postnatal 1, 3, 7, 14 and 21 days of age (n⫽3/group), (2) microglial cells cultured in the absence of LPS, (3) microglial cells cultured in the presence of LPS and (4) microglial cells cultured in the presence of LPS and RA (LPS⫹RA). Total RNA was extracted from corpus callosum or the microglial cells in culture with the RNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer’s instructions and quantified spectrophotometrically at 260 nm. For reverse transcription (RT), 2 ␮g of RNA was mixed with 1 ␮M of oligo(dT) primer (Invitrogen Life Technologies, CA, USA), incubated at 70 °C for 5 min and chilled on ice. RT solution consisting of 1 ␮l dNTP (10 mM), 5 ␮l 5⫻ cDNA synthesis buffer, 1 ␮l of RNase inhibitor and 200 U M-MLV reverse transcriptase (Promega, WI, USA), was added to the sample (total 25 ␮l) and incubated at 42 °C for 50 min. The RT reaction was stopped by heating at 95 °C for 5 min. The cDNA was used to amplify a 209-bp fragment of the IGF-I using specific primers (forward 5=-CAGTTCGTGTGTGGACCAAG-3=; reverse 5=-TCTTGGGCATGTCAGTGTG-3=) and 180-bp fragment of IGF-II (forward 5=-TCTCATCTCTTTGGCCCTTCG-3=; reverse 5=-AAGCAGCACTCTTCCACGAT-3=). RT-PCR was performed using FastStart DNA Masterplus SYBR Green I reaction mix (Roche Diagnostics, Indianapolis, IN, USA). Rat ␤-actin (285bp) was amplified as the control for normalizing the quantities of transcripts of each of the above gene using forward primer 5=-TCATGAAGTGACGTTGACATCCGT-3= and reverse primer 5=-CCTAGAAGCATTTGCGGTGCAGGATC-3=. The RT-PCR products of IGF- I

C. Kaur et al. / Neuroscience 138 (2006) 1233–1244 Table 1. Antibodies used for immunohistochemistry and double immunofluorescence labeling

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Antibody

Host

Source

Dilution

confirmed that the SYBR green bound immunofluorescence was from the amplicon. Gene expression was quantified using a modification of the 2⫺⌬⌬ct method as previously described (Livak and Schmittgen, 2001). All data are expressed as mean⫾S.D.

IGF-I

Rabbit-polyclonal

1:200

Western blotting

IGF-II IGF-IR

Rabbit-polyclonal Rabbit-polyclonal

1:200 1:50

OX-42

Rat-monoclonal

Santa Cruz Biotechnology, CA, USA Santa Cruz Biotechnology Cell Signaling Technology, MA, USA Harlan, Sera Lab, Loughborough, UK

The following materials were used for Western blot analysis: (1) supraventricular corpus callosum from brains of Wistar rats at postnatal 1, 3, 7, 14 and 21 days of age (n⫽3/group), (2) microglial cells cultured in the absence of LPS, (3) microglial cells cultured in the presence of LPS and (4) microglial cells cultured in the presence of LPS and RA (LPS⫹RA). The corpus callosum tissues were snap-frozen and homogenized with T-PER tissue protein extraction reagent (Pierce Biotechnology, CA, USA) containing cocktail protease inhibitors. Homogenates were centrifuged at 15,000⫻g for 10 min and the supernatant collected. Protein extracts from the cultures were

1:100

and IGF- II mRNA were subjected to 1.5% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light, and were captured using ChemiGene2 (Syngene, Cambridge, UK) image analyzer. The predicted size was detected and

Fig. 1. RT-PCR analysis of IGF-I and IGF-II mRNA expression in 1–21 day old rat corpus callosum. Left panel represents 1.5% agarose gel stained with ethidium bromide of RT-PCR products of the IGF-I (A) and IGF-II (B) in the corpus callosum of 1, 3, 7, 14 and 21 day old rats (P1–P21, lanes 2– 6). Lanes 1 and 7 show 100 bp fragments from a DNA ladder. Right panel shows bar graphs representing the fold changes of mRNA levels quantified by normalization to the ␤-actin as an internal control. Significant differences in the IGF-I and IGF-II mRNA are observed with increasing age (* P⬍0.05) as compared with 1 day of age.

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prepared using a protein extraction kit (Novagen, Germany, Cat. No. 71009). Protein concentrations were determined by the Bradford method (Bradford, 1976) using bovine-serum albumin (Sigma-Aldrich) as a standard. Samples of supernatants containing 20 ␮g protein were denatured at 95 °C for 5 min and were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels, in a Mini-Protein II apparatus (Bio-Rad, CA, USA). Protein bands were electroblotted onto 0.45 ␮m polyvinylindene difluoride (PVDF) membranes (Bio-Rad) at 1.5 mA/cm2 of membrane for 1 h in Towbin buffer, pH 8.3, to which 20% (v/v) methanol had been added. After transfer, the membranes were blocked with 5% (w/v) non-fat dried milk and 0.05% (v/v) Tween-20 in 20 mM Tris–HCl buffer, pH 7.6, containing 137 mM sodium chloride (TBST). The membranes were then separately incubated with dilutions of the polyclonal IGF-I and IGF-II (1:1000) antibodies in blocking solution overnight at 4 °C and then incubated with the appropriate secondary antibody, HRP conjugated anti-rabbit, 1:5000 (Amersham Biosciences, Little Chalfont, UK). Specific binding was revealed by Supersignal West Pico-chemiluminescence kit (Pierce Biotechnology) following the manufacturer’s instructions. For load control, after intensive washing, membranes were incubated with monoclonal mouse anti-actin (1:3000) (Sigma-Aldrich) and revealed as explained above. Precision prestained standards (Bio-Rad) were used as molecular weight markers. X-ray films (Amersham Biosciences) were scanned with a computer-assisted G-710 densitometer (Bio-Rad) to quantify band optical density using Quantity One software.

Immunohistochemistry Postnatal rats at 1, 3, 7, 14 and 21 days of age (n⫽4/group) were used for immunohistochemical analysis. The rats were anesthetized with 3.5% chloral hydrate and perfused with Ringer’s solution until the liver and lungs were clear of blood. This was followed by perfusion with an aldehyde fixative composed of a mixture of periodate–lysine–paraformaldehyde with a concentration of 2% paraformaldehyde. The brains were removed following the perfusion and kept in a similar fixative as above for 4 h. The tissues were then kept at 4 °C overnight in 0.1 M phosphate buffer containing 15% sucrose. Coronal frozen sections of the brains of 40 ␮m thickness at the level of optic chiasma were cut and divided into two sets. They were rinsed in phosphate-buffered saline (PBS). For blocking of non-specific antigens, the sections were pre-incubated for 30 min in PBS containing 5% normal goat serum and 0.2% Triton X-100. They were then incubated at room temperature (22 °C) with anti-IGF-I and anti-IGF-II (Santa Cruz Biotechnology, CA, USA) polyclonal antibodies (Table 1) at a dilution of 1:200. Subsequent antibody detection was carried out using biotinylated anti-rabbit IgG and Vectastain ABC kit (PK-4002, Vector Laboratories, CA, USA) with 3,3-diaminobenzidine (DAB, Sigma-5637) as a peroxidase substrate and intensified with nickel ammonium sulfate. The sections were counterstained with 1% Methyl Green, dehydrated and mounted in Permount. Sections were then examined under the light microscope and the images captured using the Image-pro plus Vision 4.1 software. For negative controls, some sections were incubated in a medium omitting the primary antibody.

Statistics For RT-PCR and Western blots, data were expressed as mean⫾S.D. A Student’s t-test was used to determine the statistical significance of differences between controls and after treatment with LPS or LPS⫹RA in the microglial cells. Results were considered as significant at P⬍0.05.

Double immunofluorescence One and 3 day old rats were used for double immunofluorescence study. The rats (n⫽3/group) were anesthetized with 3.5% chloral hydrate and perfused with an aldehyde fixative composed of a mixture of periodate-lysine-paraformaldehyde with a concentra-

Fig. 2. Western blot analysis shows the expression of IGF-I and IGF-II proteins in the corpus callosum of 1–21 day old rats. Upper panel shows the Western blotting bands detected at approximately 7.5 kDa using the IGF-I and IGF-II antibodies and ␣-actin used as an internal control. Lower panel represents bar graphs showing significant changes in the optical density (given as mean⫾S.D. of optical density) of IGF-I and IGF-II protein expression in 1, 3, 7, 14 and 21 day old rats (P1–P21). Significant differences in the IGF-I and IGF-II protein expression in various ages are observed (* P⬍0.05) in comparison to 1 day of age.

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tion of 2% paraformaldehyde. Frozen sections of the brain at 40 ␮m thickness were cut and rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 min and subsequent washing with PBS. The sections were then incubated at room temperature with a cocktail mix of two primary antibodies (IGF-I, IGF-II or IGF-IR and OX-42, Table 1). OX-42 is a specific marker for AMC which recognizes the complement type 3 receptors (CR3). Subsequent antibody detection was carried out with a cocktail mix of two secondary antibodies: CY3- conjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-mouse IgG. The sections were then washed in PBS and mounted in vector fluorescent medium (Dako Cytomation, CA, USA). Cellular colocalization was then studied in a confocal microscope (Carl Zeiss, LSM 410, Germany). Double immunofluorescence labeling was also carried out in microglial cell cultures in the presence or absence of LPS and LPS⫹RA. The cells were washed with PBS (pH 7.4) to eliminate the culture medium. They were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min, blocked in 1% bovine serum albumin (BSA) in 0.1 M PBS containing 0.1% Triton X-100 for 30 min. The cells were then incubated at 4 °C overnight with a cocktail mixture of two primary antibodies (IGF-I or IGF-II and OX-42 and processed and visualized as described above for corpus callosum).

RESULTS Analysis of IGF-I and IGF-II mRNA expression in the corpus callosum by real time RT-PCR (Fig. 1) Gene expression of IGF-I and IGF-II was detected in the corpus callosum of rats from 1 day to 21 days of age. IGF-I and IGF-II mRNA expression was significantly increased at 3 days as compared with 1 day old rats (P⬍0.05). Thereafter, both IGF-I and IGF-II mRNA declined significantly up to 21 days of age (P⬍0.05). Analysis of IGF-I and IGF-II protein expression in the corpus callosum by Western blot (Fig. 2) IGF-I and IGF-II protein expression at 7.5 kDa in the corpus callosum was detected by Western blotting. The IGF-I protein quantity increased significantly in 3, 7 and 14 day (P⬍0.05) old rats in comparison to 1 day old rat whereas the expression was not significantly different at 21 days of age. The IGF-II protein quantity increased significantly at 3 and 7 days of age but was significantly reduced at 14 and 21 days (P⬍0.05). Immunohistochemical analysis AMC in the corpus callosum above the lateral ventricle in 1 and 3 day old rats were labeled with IGF-I. The IGF-I positive cells were round (Fig. 3A) with occasional cells exhibiting stout processes. Expression of IGF-I immunoreactivity, however, was attenuated at 7 days and was diminished further at 14 and 21 days. Besides the corpus callosum, AMC in the cavum septum pellucidum and in the subventricular region of the ventricles (Fig. 3B) were also stained positively with IGF-I. AMC cells in 1 and 3 day old rats showed a positive immunoreaction for IGF-II (Fig. 3C), though its immunoreactivity appeared to be weaker as compared with IGF-I. IGF-II immunoreactivity was hardly detected in the AMC at 7 days and in older rats in the corpus callosum.

Fig. 3. Corpus callosum of a 1 day old rat showing round AMC (arrows) expressing IGF-I immunoreactivity (A). Many IGF-I positive amoeboid cells (arrows) are distributed in the subventricular zone (SVZ) of the lateral ventricle in a 1 day old rat (B). AMC (arrows) in the corpus callosum of a 3 day old rat showing weak expression of IGF-II (C). Some elongated cells (arrowheads), presumably the glioblasts are also showing IGF-II expression. Scale bars⫽50 ␮m A, B; C⫽10 ␮m.

Double immunofluorescence labeling Double immunofluorescence labeling was in agreement with the immunohistochemical results. The round immunoreactive cells that exhibited IGF-I, IGF-II and IGF-IR immunofluorescence were co-localized with OX-42 positive cells confirming them to be AMC (Fig. 4).

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Fig. 4. Confocal images showing the distribution of OX-42 (A; green) and IGF-I positive (B; red) AMC (arrows) in the corpus callosum of a 3 day old rat. The colocalized expression of OX-42 with IGF-I expressing AMC (arrows) can be seen in C. D–F are showing the expression of OX-42 (D), IGF-II (E) and OX-42⫹IGF-II colocalization (F) in the AMC (arrows). G–I show the expression of OX-42 (G), IGF-IR (H) and OX-42⫹IGF-IR (I) in the AMC (arrows). Scale bars⫽50 ␮m A–I.

LPS enhances IGF-I and IGF-II mRNA and protein expression in the activated microglia The cells prepared from primary cultures were considered as microglia as they were immunostained with the monoclonal antibody OX-42 which is a specific marker of these cells (Ling et al., 1990). The microglial cells were stimulated by treatment with LPS. The specificity of RT-PCRs was verified by checking that the PCR products were of the expected size by gel electrophoresis. The primer pair for each gene resulted in

a single product with the desired length: IGF-I (209 bp) and IGF-II (180 bp). Real-time RT-PCR showed that LPS enhanced the IGF-I and IGF-II mRNA expression in microglial cultures significantly (P⬍0.05) in comparison to cultures which were not treated with LPS (Fig. 5). IGF-I and IGF-II protein expression by Western blots also showed a significant increase (P⬍0.05) with LPS treatment (Fig. 6). Double immunofluorescence labeling showed that the expression of OX-42 on microglial cells was completely

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Fig. 5. RT-PCR analysis of expression of IGF-I and IGF-II mRNA in the microglial cells treated with LPS and LPS⫹RA. Bands of predicted size of IGF-I (209 bp) and IGF-II (180bp) were detected in the cultured microglial cells. ␤-Actin (285 bp) was used as positive control. The graph in the right panel shows IGF-I and IGF-II mRNA fold changes in the control, LPS- and LPS⫹RA-treated microglial cells. The values represent the mean⫾S.D. (n⫽3) and were normalized to the ␤-actin values. Significant differences in the IGF-I and IGF-II mRNA expression between the control and LPS treated cells as well as LPS-treated and LPS⫹RA-treated cells are observed (* P⬍0.05).

co-localized with IGF-I and IGF-II expression (Fig. 7A–C and Fig. 8A–C). Following treatment with LPS, the expression of OX-42, IGF-I and IGF-II was upregulated (Fig. 7D–F and Fig. 8D–F) in the microglial cells. Massive upregulation was observed in IGF-II expression (Fig. 8E) as compared with IGF-I (Fig. 7E). A few smaller cells, which may be less activated, showed a moderate increase in the IGF-I and IGF-II expression (Fig. 7D–F and Fig. 8D–F). RA suppresses IGF-I and IGF-II mRNA and protein expression in LPS-activated microglial cells With the addition of RA to cultures treated with LPS, the IGF-I and IGF-II mRNA expression decreased significantly (P⬍0.05) as compared with that with LPS treatment alone (Fig. 5). However, this decrease was not significant when compared with the IGF-I and IGF-II expression in cells without LPS treatment. Similarly, the protein expression

with RA treatment in the presence of LPS showed a statistically significant decline (P⬍0.05) when compared with the values with LPS treatment alone (Fig. 6). With the addition of RA to cell cultures activated with LPS (LPS⫹RA), there was a drastic reduction in the intensity of OX-42, IGF-I and IGF-II staining (Fig. 7G–I and Fig. 8G–I). The reduced OX-42 expression was localized mainly in the perinuclear region and was hardly detected in the cytoplasmic processes as observed in the control or LPS treated cells (Figs. 7G and 8G). The IGF-I and IGF-II expression followed a similar pattern as OX-42 (Figs. 7H, I and 8H, I). Interestingly the reduced expression of OX-42, IGF-I and IGF-II in RA-treated cells appeared to be attenuated to levels lower than that of the controls (Figs. 7 and 8). These results indicate that the RA inhibits not only OX-42 expression but also IGF-I and IGF-II mRNA and protein expression in the LPS-activated microglial cells.

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Fig. 6. Western blots show that RA inhibited the LPS-induced expression of IGF-I and IGF-II proteins in microglia cultures (A and B). Left panel shows the Western blotting bands of approximately 7.5 kDa using the IGF-I and IGF-II antibodies in the control, LPS- and LPS⫹RA-treated microglia cell cultures. Graph in B shows the optical density of the IGF-I and IGF-II protein expression in the control; LPS- and LPS⫹RA-treated microglial cells. The values represent the mean⫾S.D. (n⫽3). Significant differences between the LPS- and LPS⫹RA-treated cell cultures are observed (* P⬍0.05).

DISCUSSION The present study has shown IGF-I and IGF-II mRNA and protein expression in the corpus callosum of 1 day to 21 day old rats. This period of development has been regarded as a critical period of brain development in rodents (Lee et al., 1996) during which myelination process begins and progresses (Hamano et al., 1998). IGF-I and IGF-II mRNA showed an increase at 3 days of age following which there was a steady decrease with age at 7, 14 and 21 days. Protein expression of IGF-I and IGF-II by Western blotting showed an increase up to 14 days and 7 days respectively as compared with 1 day of age. A previous study has reported that during CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines at birth to values found in adult brain (Rotwein et al., 1988). Although these authors examined various parts of brain like the olfactory bulb, cerebellum, cerebral cortex and hippocampus for IGF-I and IGF-II expression, specific regions like the corpus callosum or other white matter tracts were not examined. Our study is the first study to report the IGF-I, IGF-II and IGF-IR expression in the developing corpus callosum. At the cellular level, expression of IGF-I and IGF-II was localized in the AMC in the corpus callosum. AMC are readily distinguished from the other glial cells in the corpus callosum and subependymal region of the brain in various ages based on their staining with the antibody OX-42 and as described by us earlier (Ling et al., 1990). The present study has shown for the first time that AMC express IGF-I immunoreactivity in the developing brain. The expression of IGF-I immunoreactivity was intense in the early postnatal period but it decreased at 7 days and was negligible in 14 and 21 day old rats. This paralleled with the fact that the IGF-I mRNA in the corpus callosum was gradually decreased up to 21 days as compared with early postnatal stage i.e. 1 and 3 days.

AMC are macrophagic cells which phagocytose debris of degenerating cells and axons in the developing brain as well as exogenously administered substances like horseradish peroxidase and rhodamine isothiocyanate (Kaur et al., 1986; Xu et al., 1993). Recently we have shown that these cells can phagocytose Escherichia coli (E. coli) following intracerebral injections of the live bacteria (Kaur et al., 2004). As phagocytosis is one of the primary functions of AMC which is related to the state of activation of these cells, it is suggested that IGF-I expression in these cells may be linked to the state of activation of the cell. IGF-I has been shown to enhance phagocytic activity of neutrophils in vitro when they were challenged with E. coli (Balteskard et al., 1998). Inoue et al. (1995) have demonstrated that IGF-I increases the killing capacity and phagocytosis of peritoneal macrophages when they were activated by E. coli. In this connection, the present in vitro results have shown that LPS-induced activation of microglial cells is associated with a significant increase in IGF-I mRNA and protein expression in these cells. A variety of inflammatory mediators has been reported to induce expression of IGF-I in macrophages (Arkins et al., 1995). Tumor-necrosis factor-␣ (TNF-␣) is one such factor which stimulates the production of IGF-I by macrophages (Fournier et al., 1995). Since LPS-induced activation of microglial cells is associated with induction of increased levels of TNF-␣ (Dheen et al., 2005), this may be one of factors leading to elevated expression of IGF-I in LPS stimulated microglial cells. In this connection, a previous study has shown a reduced expression of insulin-like growth factor binding proteins (IGFBPs) in LPS activated microglia (Chesik et al., 2004). As IGFBPs regulate the IGF-I expression, it was suggested by these authors that their downregulation following microglial activation facilitates the autocrine/paracrine actions of IGF-I to stimulate microglial proliferation (Chesik et al., 2004).

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Fig. 7. Confocal images of cultured microglia showing the expression of OX-42 (A, green), IGF-I (B, red) and co-localized expression of OX-42 and IGF-I (C). D–F show the expression of OX-42 (D, green), IGF-I (E, red) and colocalized expression of OX-42 and IGF-I (F) after treatment with LPS. Note the elevated expression of IGF-I following LPS treatment (E) as compared with control cells (B). G–I show the expression of OX-42 (G, green), IGF-I (H, red) and colocalized expression of OX-42 and IGF-I (I) in LPS⫹RA-treated cells. The expression of IGF-I is greatly reduced after the addition of RA (H). Scale bars⫽50 ␮m A–I.

RA, which has an anti-inflammatory role (Choi et al., 2005), attenuates the production of inflammatory media-

tors (Mehta et al., 1994) such as TNF-␣ and inducible nitric oxide (iNOS). TNF-␣ production by macrophages acti-

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Fig. 8. Confocal images of cultured microglia showing the expression of OX-42 (A, green), IGF-II (B, red) and co-localized expression of OX-42 and IGF-II (C). D–F show the expression of OX-42 (D, green), IGF-II (E, red) and colocalized expression of OX-42 and IGF-II (F) after treatment with LPS. Note the elevated expression of IGF-II following LPS treatment (E) as compared with control cells (B). G–I show the expression of OX-42 (G, green), IGF-II (H, red) and colocalized expression of OX-42 and IGF-II (I) in LPS⫹RA-treated cells. The expression of IGF-II is greatly reduced after the addition of RA (H). Scale bars⫽50 ␮m A–I.

C. Kaur et al. / Neuroscience 138 (2006) 1233–1244

vated by endotoxin is completely inhibited by RA treatment (Mehta et al., 1994). The present results have shown that LPS-induced IGF-I mRNA and protein expression was significantly reduced with RA treatment. Along with suppressing the production of inflammatory mediators as reported previously (Mehta et al., 1994; Dheen et al., 2005), we have shown in the present study that RA suppresses the IGF-I expression. This suppression may be as a result of direct action of RA on IGF-I expression or through its suppressive action on inflammatory mediators such as TNF-␣. The inhibitory effects of RA on microglial activation in vitro have been reported to be mediated probably via the retinoic acid receptor ␤ (Dheen et al., 2005). A noteworthy feature in RA-treated microglial cells was that the expression of IGF-I and IGF-II was reduced to levels that appeared to be even lower than that of the controls. Very interestingly, RA also markedly suppressed OX-42 expression in the microglial cells indicating reduced CR3-mediated phagocytosis. It is suggested that the concomitant reduction in IGF-I and IGF-II expression by RA is linked to inhibition or redistribution of CR3 marked by OX-42. It may be significant to note that OX-42 immunoreactivity appeared to be localized preferentially in the perinuclear area in RA-treated microglial cells. That LPSchallenged microglial cells treated with RA exhibit lower OX-42, IGF-I and IGF-II compared with controls suggests that RA is an extremely potent inhibitor for activated microglia as was reported recently by us (Dheen et al., 2005). From the results of the present investigation, it is reasonable to suggest that IGF-I may be involved in enhancing the activity of these cells in inflammatory conditions. The expression of IGF-I was weak in the AMC in 7 days old rats and was not observed in older rats. This could be related to their transformation into ramified microglial cells which are considered as resting cells (Ling and Wong, 1993; Kaur et al., 2001). IGF-I is also known to stimulate the proliferation of immunocompetent cells and modulate cellular immune functions like natural killer cell activity and oxidative burst besides phagocytosis and killing capacity of neutrophils and macrophages (Auernhammer and Strasburger, 1995). In view of the above, IGF-I may also be related to antigen presenting function of AMC as these cells express antigens of the major histocompatibility complex class I (MHC I) and, hence, can participate in a possible immune response (Ling et al., 1991). Our previous studies have also shown induction of MHC II antigens in the AMC following administration of live E. coli in the corpus callosum (Kaur et al., 2004). The functional significance of expression of IGF-II immunoreactivity in AMC in 1 and 3 day old rats also remains speculative. Like IGF-I, it may also be related to the state of activation of the cells for their phagocytic and immune functions. This is supported by the enhanced IGF-II mRNA and protein expression in LPS-activated microglial cells. IGF-I and IGF-II expression in the AMC may also have paracrine functions such as modulation of the proliferation and development of the oligodendrocytes and myelination. IGF-I is known to be important for oligodendrocyte survival

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and myelination (Guan et al., 2001). Ye et al. (2002) have reported that myelination during early development is altered in the absence of IGF-I by mechanisms that involve a reduction in oligodendrocyte proliferation and that IGF-II could compensate partly for the actions of IGF-I on myelination. In this connection, a role for microglia/macrophages in active myelination in the developing brain has been proposed (Hutchins et al., 1992); macrophage-derived growth factors being involved in myelinogenesis and myelin repair in inflammatory demyelinating disease (Loughlin et al., 1997). IGF-I mRNA and protein expression levels have been found to be increased in the corpus callosum during the recovery phase in experimentally induced demyelination (Mason et al., 2001). This correlated with a large number of IGF-I positive microglia/macrophages indicating that these cells produce IGF-I in demyelinating lesions which plays a role in oligodendrocyte repopulation and remyelination (Mason et al., 2001). The actions of IGF-I and IGF-II appear to be mediated primarily by their interactions with the IGF-IR as IGF-IR positive cells were co-localized with OX-42 positive AMC. The present results have shown that IGF-I and IGF-II are constitutively expressed by AMC during development. The expression, however, is downregulated once the cells transform into ramified microglia in the second week of postnatal life. The expression of IGF-I and IGF-II is enhanced by treatment with LPS and attenuated with concomitant treatment with RA that also suppresses CR3 expression marked by OX-42. Acknowledgments—This study was supported by a research grant (R181-000-065-112) from the National University of Singapore.

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(Accepted 1 December 2005) (Available online 31 January 2006)