key: cord-0007775-pq8dkp37
authors: Suzumura, Akio; Sawada, Makoto; Itoh, Yasuhiro; Marunouchi, Tohru
title: Interleukin-4 induces proliferation and activation of microglia but suppresses their induction of class II major histocompatibility complex antigen expression
date: 2002-11-13
journal: J Neuroimmunol
DOI: 10.1016/0165-5728(94)90031-0
sha: edeac2115b5a6689c57a5fa62a1b7330bfb5eb8b
doc_id: 7775
cord_uid: pq8dkp37

We recently found that microglia, brain macrophages, express interleukin-4 (IL-4) receptor mRNA in vitro. Since IL-4 exhibits a variety of functions on the cells of monocyte-macrophage lineage, we examined the effects of IL-4on the functions of microglia. Recombinant IL-4 induced the proliferation of microglia in a dose- and time-dependent manner as determined by MTT colorimetric assay, [(3)H]thymidine uptake and bromodeoxyuridine (BrdU) incorporation. IL-4 also synergistically enhanced the proliferation of microglia with such colony-stimulating factors as IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). It also increased acid phosphatase activity and superoxide anion formation by these cells. Despite these positive effects on proliferation and activation, IL-4 suppressed the IFN γ-induced class II MHC antigen expression in these cells. Since these effects of recombinant IL-4 inhibited by the addition of monoclonal antibody against IL-4 receptors, the effects of IL-4 on microglia appear to be a specific function via IL-4 receptors. Although microglia and astrocytes produce a variety of immunoregulatory cytokines, neither cell produced IL-4 as determined by bioassay or detection of IL-4 mRNA by RT-PCR method. Thus, the exogenous IL-4 may contribute to the accumulation of microglia in or around inflammatory lesions in the central nervous system, and may be involved in the regulatory mechanisms of microglia.

Microglia are one of the three types of glial cells in the central nervous system (CNS). Although their origin is not clear, these cells appear to be from the monocyte-macrophage lineage, and enter the CNS late in the embryonic stage (Del Rio-Hortega, 1932; Imamura et al., 1990) . Their phenotypic or functional characteristics are similar to those of monocyte-macrophages (Austin and Gordon, 1981; Perry et al., 1985; Giulian and Baker, 1986; Suzumura et al., 1987) . Their growth and functions are regulated by colony-stimulating factors as are the monocyte-macrophages Suzumura et al., 1990; . They produce such monokines as IL-1, IL-6 and tumor necrosis factor (TNF)a (Giulian and Baker, 1986; Sawada et al., 1989 Sawada et al., , 1992 . In addition, microglia can be induced to express class II major histocompatibility complex (MHC) antigens (Suzumura et al., 1987) , and reportedly present antigens to T cells in an antigen-specific manner (Righi et al., 1989) . Because of their similarity to macrophages, microglia are considered to play a key role in the development of various CNS diseases as inflammatory or immunoregulatory cells. In the case of injury, inflammation or neuronal degeneration, they appear early at the site where they seem to proliferate (Ohmori et al., 1992) . An increase in number of microglia leads to the formation of clumps of small nuclei called 'glial star', which is a main histological feature of neuronal loss or CNS tissue damage (Duchen, 1992) . Factors in inflammatory prodficts may perhaps induce the proliferation of microgli a in these disorders.

IL-4 was initially identified as a B cell growth factor (Haward et al., 1982) , being designated as B cell growth factor 1 (Lee et al., 1986) . It is a potent B cell growth factor , a major regulator of the IgG1 and IgE isotype by activated B cells (Coffman et al., 1985; Sideras et al., 1985; Vitetta et al., 1985) , and the primary inducer of the low affinity IgE receptor (CD23) on B cells (Defrance et al., 1987) . Besides these effects on B cells, IL-4 exerts a regulatory function on macrophages. It activates the tumoricidal function of macrophages (Crawford et al., 1987) and enhances the antigen-presenting cell functions of certain types of macrophage (Zlontnik et al., 1987) . However, a suppressive effect of IL-4 on macrophages has also been reported. It suppresses formation of macrophage colonies by M-CSF, and also suppresses the production of monokines by these cells (Essner et al., 1989; Hart et al., 1989; Gautam et al., 1992) . It is therefore possible that IL-4 may influence the functions of microglia as well. Recent studies suggest that IL-4 is involved in the pathophysiology of CNS disorders. IL-4 mRNA has been detected in the CNS in the acute phase of viral infections (Moskophidis et al., 1991) , toxoplasmic encephalitis (Hunter et al., 1992) , experimental allergic encephalomyelitis (Kennedy et al., 1992) and multiple sclerosis (Wucherpfennig et al., 1992) .

A variety of immunoregulatory cytokines are produced in the CNS which affect functions of neural cells as autocrine or paracrine mediators, and form a unique network of cytokines (Sawada et al., 1989 (Sawada et al., , 1992 Lieberman et al., 1989; Ohno et al., 1990) . However, whether IL-4 is produced in the CNS, or whether it has any effect on neural cells is not known. We recently found that isolated microglia express IL-4 receptor mRNA . In this study, we examined the production of IL-4 by glial cells, and examined the effects of IL-4 on the various functions of the isolated microglia in vitro.

Recombinant murine IL-3, IL-4, GM-CSF, IFN3, and human M-CSF, and rat monoclonal antibody against murine IL-4 receptors (IL-4R) were obtained from Genzyme (Boston, MA). Rat monoclonal antimurine IL-5 antibody was obtained from Endogen (Boston, MA) and used for the control of the IL-4R antibody. The monoclonal antibody against glial fibrillary acidic protein (GFAP) was; a gift of Dr. S.U. Kim (British Columbia, Canada). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and Nitroblue tetrazolium (NBT) were purchased from Sigma (St. Louis, MO). Lipopolysaccharide (LPS; from Escherichia coli) was purchased from Difco (Detroit, MI).

Primary mixed glial cell cultures were prepared as described previously (Suzumura et al., 1984) , using newborn C3H/HeN mice (Charles River Japan, Shizuoka, Japan). In brief, after the meninges were carefully removed, the brain was dissociated by passing it through a 320-/zm pore nylon mesh. After being washed with Hanks' balanced salt solution, the cell suspension was triturated and plated in 75-cm 2 culture flasks (Falcon 3024, Beckton-Dickinson, N J) at a density of two brains/flask in 10 ml Eagle's MEM supplemented with 10% fetal calf serum, 5/~g/ml bovine insulin, and 0.2% glucose. Microglia were isolated on the 14th day by the 'shaking off' method as previously described (Suzumura et al., 1987) . The purity of the cultures was 97-100% as determined by immunostaining with Mac 1 antibody (Hybritech, San Diego, CA, used in 1:50 dilution) Suzumura et al., 1990 Suzumura et al., , 1991 . Astrocyte-enriched cultures were prepared from the primary mixed glial cell cultures by repetitive trypsinization and replating (Suzumura et al., 1988) . The purity of the cultures exceeded 95% as determined by indirect immunofluorescence staining with anti-GFAP antibody (Suzumura et al., 1988) .

The expression of mRNA for IL-4 and IL-4R was studied by the RT-PCR method as described below. A T cell line, D10N3 (provided by Dr. K. Onozaki, Nagoya City University, Japan), was used as a positive control for the expression of IL-4 mRNA. IL-4 activity in the supernatant was assessed by a bioassay using an IL-2and IL-4-dependent cell line, CTLL-2, and the MTT colorimetric assay (Sawada et al., 1992) . Microglia and astrocyte-enriched cultures were plated in 2.5-cm diameter culture dishes (Falcon 3001, Beckton-Dickinson, Lincoln Park, NJ) at a density of 5 X 105/ml for 24 h with or without 1 Izg/ml LPS or 100 U/ml IFN3,. The supernatant was then collected and stored at -70°C until assayed for the IL-4 activity. After remaining cells had been washed three times with PBS, the total RNA was isolated from the cells using the method of Chomczynski and Sacchi (1987) . An amount of 1 ~g of RNA was used for first-strand cDNA synthesis at 37°C for 90 min with 50 U of recombinant M-MuLV reverse transcriptase (Boehringer-Mannheim, Germany) and 0.2 /zg of DNA random hexamers. The reaction was terminated by boiling for 5 min, and 2 ~1 of the mixture was amplified by 30 PCR cycles with 0.5 units of Taq polymerase (Promega, Madison, WI) and 0.5 tzg each of the sense and antisense primers. The thermal cycle profile was as follows: 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. An aliquot (5 tzl) of the PCR mixture was subjected to electrophoresis in 2% agarose gel. The agarose gel was then stained with ethidium bromide and photographed. The primers used are; IL-4 sense, 5'-ATGG-GTCTCAACCCCCAGCTAGT, IL-4 antisense, 5'-GCTCTTTAGGCTTTCCAGGAAGTC, IL-4R sense, 5'-CCAGTGGTAATGTGAAGC, IL-4R antisense, 5'-GACAAGTITTCCAGTGCG.

MTT is cleaved by living cells to yield a formazan product. Thus, the measurement of a formazan product using a colorimetric method allows estimation of the relative number of surviving cells. Microglia were plated into wells of a 96-well microtiter plate (Falcon 3072, Beckton-Dickinson, Lincoln Park, N J) at a density of 2 x 104/100/xl culture medium, and were stimulated with graded concentrations (1-100 U/ml) of IL-4 for 72 h. To examine the time course, microglia were stimulated as above for 24-96 h. An amount of 50 /zg/ml MTT solution was added to each well in the final 6 h. The reaction was then halted by adding 150 /zl of 0.04 N HCl-isopropanol. After sonication, the optical density (OD620nm) of the solubulized materials was measured by an Immunoreader J200 (Inter Med Japan, Tokyo, Japan). In some experiments, graded concentrations of monoclonal antibody against IL-4 receptors (final dilution 1 : 102-1:106) were applied with the stimulants, using the same dilution of rat monoclonal anti-mouse IL-5 antibody as a control.

Microglia were plated into wells of a 24-well culture plate (Falcon 3047, Beckton-Dickinson, Lincoln Park, NJ) at a density of 1 x 105/500 /zl, and were stimulated with 0.1-100 U/ml IL-4 in the presence or absence of 20 U/ml IL-3, 20 U/ml GM-CSF or 10 U/ml M-CSF for 72 h. 1/zCi/well [3H]thymidine was added in the final.12 h. The cultures were then washed three times with PBS. Cells were lysed with 100 #l of 0.2 N NaOH and were precipitated with 20/zl of 50% trichloroacetic acid (TCA). After extensive washing with 5% TCA, the precipitates were collected on glass fiber filter paper. The radioactivity of each paper was counted in a liquid scintillation counter (Beckman, USA).

Parallel cultures were plated onto 14-mm glass coverslips at a density of 5 x 104/ml, and stimulated with the same reagents as above for 72 h. BrdU (10/.~g/ml) was added for the final 12 h. Coverslips were then immunolabeled with FITC-labeled anti-BrdU antibody (Beckton-Dickinson, Lincoln Park, N J, final dilution 1 : 3). FITC-positive BrdU-incorporated microglia were counted under a fluorescence microscope (Zeiss, Germany), as described (Suzumura et al., , 1991 .

Microglia were plated into wells of a 96-well microtiter plate at a density of 2 x 104/100 /xl culture medium. They were cultured with graded concentrations of IL-4 (1-100 U/ml). After 3 days in culture, acid phosphatase activity and superoxide anion formation (NBT-reducing activity) were assessed as described previously . Results were expressed as mU/well of acid phosphatase activity and nmol formed NBT diformazan for superoxide anion formation Suzumura et al., 1990) . All the experiments were done in at least quadruplicate, and the t-test was used for the statistical analyses. We also evaluated the effects of IL-4 on the induction of class II MHC antigen expression by IFN7. For this purpose, microglia were plated in a 2.5-cm culture dish at a density of 5 x 105/ml culture medium, and stimulated with 100 U/ml IFN7 in the presence or absence of 1-100 U/ml IL-4. In some experiments, anti-IL-4R antibody (1 : 103 in final dilution) was added with IFN7 and IL-4. After 12 h in culture, they were washed three times with PBS. Total RNA was isolated and the expression of mRNA for class II MHC antigens was examined by the RT-PCR method described above. The following primer was used to detect mRNA for I-Ak-a class II MHC; sense, 5'-AAGAAGGA-GACTGTCTGGATGC, antisense, 5'-TGAATGAT-GAAGATGGTGCCC (Bishop et al., 1988) . Microglia were also examined for their surface expression of MHC class II antigens by indirect immunofluorescence and flow cytometric analyses. For this, microglia were stimulated with 100 U/ml IFN7 for 48 h either in the presence or absence of 100 U/ml IL-4. Non-stimulated cultures were used for the control of the effects of IFN7 or IL-4. They were then stained with the mouse monoclonal anti-Ia k antibody (Meiji Nyugyo, Tokyo, Japan, 1 : 50 dilution) and FITC-conjugated anti-mouse IgG antibody (Cappel, West Chester, PA, 1:50 dilution), using the monoclonal anti-la d antibody (Meiji Nyugyo, Tokyo, Japan, 1 : 50 dilution) as the control of the first antibody. The samples were analyzed on an EPICS Elite (Coulter, Hialeah, FL).

CTLL-2, known as an IL-2-dependent cell line, was also induced to grow in response to IL-4 as shown in DIO microglia astrocyte 1 2 3 the previous paper (Gearing et al., 1991; Mosmann and Fong, 1989) . However, the supernatant from astrocytes and microglia, either stimulated or unstimulated, did not induce the growth of CTLL-2, indicating the absence of a sufficient amount of IL-4 as well as that of IL-2 in the supernatant. No mRNA for IL-4 was detected in the glial cells, while it was detected in D-10N3 cell line used as the positive control (Fig. 1) . However, mRNA for IL-4 receptor was detected in the microglia revealed two bands of the predicted sizes; 328 and 240 bp (Fig. 2, lane 3) .

The microglia treated with 1-100 U/ml IL-4 for 72 h increased in number; this effects of IL-4 was neutralized by adding anti-IL-4 receptor antibody (Fig. 3) , but not by anti-IL-5 antibody used as a control. The mitogenic effects of IL-4 were time-and dose-dependent when assessed by MTT-colorimetric assay (Fig. 4) . The effects of each dose of IL-4 were neutralized by adding anti-IL-4 receptor antibody in a dose-dependent manner (Fig. 5) . Incorporation of [3H]thymidine was also Microglia were treated with 1-300 U/ml IL-4 for up to 4 days. At 1, 2, and 3 days after stimulation, cell growth was measured by MT~I ' colorimetric assay. Fold increase was calculated as (OD620 in stimulated culture)/(OD620 in unstimulated culture).

increase by doses of IL-4 (Fig. 6 ). As we previously reported, GM-CSF and M-CSF induce the proliferation of microglia (Suzumura et al., , 1991 . In the present study the mitogenic effect of IL-4 was additive to the effects each of GM-CSF, M-CSF and IL-3 ( increased the number of BrdU-positive microglia (P < 0.001) as compared to those in unstimulated microglia (Fig. 7) , while only 100 U/ml of IL-4 induced a statistically significant increase (P < 0.001) in the GM-CSF- stimulated group, as compared to the non-IL-4-stimulated control in each group. Again, IL-4 acted synergistically with GM-CSF on the proliferation of microglia in vitro (Fig. 7) . These BrdU-positive microglia were usually ameboid (Fig. 8) . Some of these IL-4-stimulated microglia were multinucleated (Fig. 8C) . About one-third of these multinuclei were labeled with anti-BrdU antibody (data not shown).

IL-4 dose-dependently increased acid phosphatase activity and superoxide anion formation (NBT-reducing activity) of the microglia (Fig. 9) . These effects of IL-4 on microglia were almost completely neutralized by the addition of anti-IL-4 receptor antibody (used in 1 : 10 3 dilution) (Fig. 9) . Despite the positive effects of IL-4 on the activation of microglia, IL-4 suppressed the induction of class II MHC antigen expression by IFNy (Fig. 10) . The fluorescence histogram of controls, either IFNy-stimulated cultures or IFNy + IL-4stimulated culture stained with a control antibody (anti-Iad), were almost identical to that of non-stimulated culture stained with an appropriate anti-Ia k antibody (Fig. 10, left) lated microglia. Class II MHC antigen expression on microglia was induced in most cells when treated with 100 U/ml IFNy (Fig. 10) . However, the addition of 100 U/ml IL-4 decreased the expression of class II MHC antigens. The suppression observed with IL-4 was almost completely neutralized by the addition of anti-IL-4 receptor antibody (data not shown). Doses of 10-100 U/ml IL-4 effectively suppressed the class II MHC antigen expression induced by 100 U/ml IFNy (data not shown). This suppression of the induction of class II MHC antigen expression was confirmed by the decrease in mRNA expression (Fig. 11) . Unstimulated microglia did not express class II MHC antigen mRNA • acid phosphatase(mU) 0 I 10 !00

IL-4(U/ml) Fig. 9 . Acid phosphatase activity and superoxide anion formation in microglia treated with indicated doses of IL-4. Columns indicate mean values of acid phosphatase activities (mU/ml) and superoxide anion formation expressed as nmol of formed NBT diformazan. Bars indicate standard deviations (n = 9). * Significant increase (P < 0.001) as compared to unstimulated control. ** significant increase (P < 0.001) as compared to the culture stimulated with 10 U/ml IL-4.

( Fig. 11, lane 1) . IFN T induced the expression of class II MHC antigen mRNA, while the addition of 10-100 U/ml IL-4 suppressed it. Addition of anti-IL-4R antibody (1 : 103 final concentration) reversed this suppressive effect of IL-4. IL-4 itself did not induce class II MHC antigen mRNA in microglia (Fig. 11) .

This study demonstrated that microglia express functional IL-4 receptors, but not produce IL-4. Receptors for IL-4 are expressed by various types of c::,:,,xl I: i;,i i ~/ :\ ! 1 10 100 t.. d~ hematopoietic and non-hematopoietic cells Lowenthal et al., 1988) . In the neural cells, the presence of IL-4 receptors on a neuroblastoma cell line or a brain stroma cell line has been shown by receptor-ligand binding analysis (Lowenthal et al., 1988) . However, it was not known which type of cells in the CNS express IL-4R, and whether or not these receptors function. It has recently been shown that IL-4 affects the survival of hippocampal neurons in culture (Araujo and Cotman, 1993) . Since the number of GFAP-positive astrocytes and Mac 1-positive microglia was also increased by the IL-4 treatment, these authors suggested that the neurotrophic effects of IL-4 may be indirect via factors elaborated by glial cells (Araujo and Cotman, 1993) . IL-4 has been shown to increase the expression of nerve growth factor (NGF) mRNA and the secretion of NGF by astrocytes (Awatsuji et al., 1993) . It has also been shown that IL-4 suppresses TNFa and nitric oxide production by activated microglia (Chao et al., 1993) . These studies strongly suggest an effect of IL-4 on the glial cells and the presence of IL-4R on such cells. More recently, in an investigation of the expression of mRNA for various cytokine receptors in isolated glial cells and neuronal cell line by the RT-PCR method, we found that microglia, but not the neuronal cell lines, expressed the IL-4 receptor mRNA in vitro . Recombinant IL-4 induced the proliferation of microglia as determined by MTT colorimetric assay, [3H]thymidine uptake and BrdU incorporation. Since the mitogenic effects of IL-4 was neutralized by adding anti-IL-4 receptor monoclonal antibody, IL-4 seemed to exert its action by binding with the IL-4 receptors on microglia. As we demonstrated previously Suzumura et al., 1990 Suzumura et al., , 1991 , microglia respond to such colony-stimulating factors as GM-CSF and M-CSF and proliferate in vitro. These cytokines non-stimulated IFNT+IL-4 .IFN?

Class IT MHC (fluorescence intensity) are the only ones reported to induce the proliferation of microglia in vitro. IL-4 synergistically increased the proliferation of microglia with these colony-stimulating factors. In human monocyte-macrophages, IL-4 reportedly suppressed the mitogenic effects of GM-CSF, but not those of M-CSF, although IL-4 itself did not induce proliferation in these cells (Akagawa and Takasuka, 1992) . Since microglia and astrocytes do not produce IL-4 in vitro, and since IL-4 mRNA is not detected in the normal brain (Wucherpfennig et al., 1992) , the IL-4-induced proliferation of microglia may occur during an interaction with the invading T cells. Under these conditions, astrocyte-derived M-or GM-CSF may act synergistically with IL-4 to induce further proliferation of microglia. Although the M-or GM-CSF-induced proliferating microglia are rod-shaped, as we have shown previously (Suzumura et al., , 1991 , the IL-4-induced proliferating microglia were ameboid and sometimes had multiple nuclei (Fig. 8 ). An increased number of elongated rod-shaped microglia are observed in certain CNS infectious diseases (Schmidt and Gonyea, 1976) , while numerous ameboid microglia are observed in CNS inflammatory lesions. Microglia nodules or increased number of multinucleated giant microglia have been observed in patients with viral encephalitis or those with AIDS-dementia complex (Sharer, 1992) . In these inflammatory lesions, IL-4 may influence the accumulation of ameboid microglia or the formation of multinucleated cells. Indeed, IL-4 activity has been detected in the cerebrospinal fluid of animals with acute lymphocytic choriomeningitis virus infection (Moskophidis et al., 1991) . IL-4 mRNA has been detected in the lesions of acute experimental allergic encephalomyelitis (Kennedy et al., 1992) as well as those of acute multiple sclerosis, but not in chronic lesions of the disease (Wucherpfennig et al., 1992) . It is therefore possible that IL-4 plays a role in the proliferation of microglia in inflammatory lesions in the CNS. Microglia exhibit a variety of functions on the development of pathological conditions in the CNS either as inflammatory cells or immunoregulatory cells. They may play a critical role on the development of immunemediated inflammatory lesions in the CNS as antigenpresenting cells (Hickey and Kimura, 1988; Righi et al., 1989) , and function as effector cells by secreting soluble factors including cytokines (Giulian, 1987; Sawada et al., 1989) . Cytokines can influence microglial functions. IFNy induces the expression of class II MHC antigen expression in microglia (Suzumura et al., 1987) and induces antigen-presenting activities (Righi et al., 1989) . IL-1 reportedly induces IL-6 synthesis in microglia of the human embryo (Sebire et al., 1993) . M-CSF activates acid phosphatase activity and superoxide anion formation by these cells . In this study, we showed that IL-4 enhances acid phosphatase activity and superoxide anion formation, but suppressed the expression of class II MHC antigens induced by IFN7. These findings suggest that IL-4 may upregulate the functions of microglia as inflammatory cells but suppress the functions as immunoregulatory cells. Although the precise functions of IL-4 on microglia in the development of disease require further elucidation, since IL-4 was detected only in acute CNS lesions (Moskophidis et al., 1991; Hunter et al., 1992; Kennedy et al., 1992; Wucherpfennig et al., 1992) , it is possible that IL-4 may influence the acute phase of inflammatory CNS lesions by affecting microglial functions. These effects of IL-4 on activities of cells of monocytic lineage may differ according to the species or the type of cells, since human IL-4 reportedly inhibits the production of superoxide, but enhances the acid phosphatase activity of human mononuclear phagocytes (Abramson and Gallin, 1990) .

IL-4 reportedly upregulates the expression of class II MHC antigen in murine peritoneal macrophages in a manner different from that of IFN~/ (Cao et al., 1989) . However, in our experiments, IL-4 did not induce class II MHC antigens on naive microglia, and suppressed the IFNy-induced class II MHC antigen expression in microglia. Since microglia do not usually express class II MHC antigens in unstimulated cultures (Suzumura et al., 1987) or the normal brain in vivo (Imamura et al., 1990) , they may respond to IL-4 in a different manner than macrophages which constitutively express class II MHC antigens. Our results seem to be compatible with the current findings which suggest that T helper ceils 1 and 2 and their secretory products may function in a distinct fashion on macrophages to either promote or suppress nonspecific inflammatory functions. IL-4, acting synergistically with IL-10 or TGFfl, inhibits the IFNy-induced production of reactive nitrogen oxides by peritoneal macrophages, and also suppresses the killing of schistosomula by IFNy-treated macrophages (Oswald et al., 1992) . IL-4 also suppressed the IFNy-and/or IL-2-induced expression of the cytokine gene in murine peritoneal macrophages (Gautam et al., 1992) . We previously showed that TGF/3 suppresses the IFNy-induced expression of class II MHC antigen in microglia . In contrast to IL-4, TGF/3 suppresses all microglial functions, including enzyme activity, cytokine production, and CSF-induced proliferation in vitro. Thus, IL-4 may be another negative regulator in the CNS cytokine network that regulate the functions of microglia differently as compared with TGF/3.

IL-4 inhibits superoxide production by human mononuclear phagocytes

Effects of interlet~kin 4 on growth and differentiation of human monocytes

Trophic effects of interleukin-4, -7, and -8 on hippocampal neuronal cultures: potential involvement of glial derived factors

F4/80, a monoclonal antibody directed against the mouse macrophage

Interleukin-4 and -5 as modulators of nerve growth factor synthesis/secretion in astrocytes

Signaling to a B-cell clone by E-k, but not A-k, does not reflect alteration of A-k genes

Differential regulation of class II MHC determinants on macrophages by IFN3, and IL-4

Neuroprotective role of IL-4 against activated microglia

Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide activated B cells

B cell stimulatory factor-1 (interleukin-4) activates macrophages for increased tumoricidal activity and expression of Ia antigens

Human recombinant IL-4 induces Fe receptors (CD23) on normal human B lymphocytes

Cytology and Cellular Pathology of the Nervous System. Hocker

General pathology of neurons and neuroglia

IL-4 down-regulates IL-1 and TNF gene expression in human monocytes

IL-4 suppresses cytokine gene expression induced by IFN3~ and/or IL-2 in murine peritoneal macrophages

Biological and immunological assays for cytokines

Ameboid microglia as effectors of inflammation in the central nervous system

Characterization of ameboid microglia isolated from developing mammalian brain

Potential antiinflammatory effects of interleukin 4: Suppression of human monocyte tumor necrosis factor tr, interleukin 1 and prostaglandin E2

Perivascular microglia cells of the CNS are bone marrow-derived and present antigen in vivo

Identification of a T cell-derived B cell growth factor distinct from interleukin 2

Kinetics of cytokine mRNA production in the brains of mice with progressive toxoplasmic encephalitis

Generation and characterization of monoclonal antibodies against rat microglia and ontogenic distribution of positive cells

Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery

Isolation and characterization of a mouse interleukin cDNA clone that express B-cell stimulatory factor 1 activities and T-cell-and mast-cell-stimulating activities

Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus

Expression of high affinity receptors for murine interleukin 4 (BSF-1) on hematopoietic and nonhematopoietic cells

Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion on IL-6 but not IL-4, IL-5 and gamma interferon

Specific assays for cytokine prodction by T cell

High affinity receptors for B cell stimulatory factor-1 (interleukin-4) expressed on lymphocytes and other cells of hematopoietic lineage

In situ demonstration of proliferating cells in the rat central nervous system during experimental autoimmune encephalomyelitis. Evidence suggesting that most infiltrating T cells do not proliferate in the target organ

Production of granulocyte/macrophage colony stimulating factor by cultured astrocytes

IL-10 synergizes with IL-4 and transforming growth factor-/3 to inhibit macrophage cytotoxic activity

B cell stimulatory factor-1/interleukin 4

Immunohistochemical localization of macrophage and microglia in the adult and developing mouse brain

Monokine production by microglial cell clones

Production of tumor necrosis factor-alpha by microglia and astrocytes in culture

Activation and proliferation of isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C

TNFa induces IL-6 production by astrocytes but not by microglia

Expression of cytokine receptors in cultured neuronal and glial cells

Neurosyphilis

In vitro production of IL-6, IL-lbeta and tumor necrosis factor-alpha by human embryonic microglia and neural cells

Pathology of HIV-1 infection of the central nervous system: A review

Partial biochemical characterization of IgGl-inducing factor

The isolation and long term culture of oligodendrocytes from newborn mouse brain

MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon

Induction of glial cell MHC antigen expression in neurotropic corona virus infection: characterization of the H-2 inducing soluble factor elaborated by infected brain cells

Effects of colony stimulating factors on isolated microglia in vitro

Morphological transformation of microglia in vitro

Transforming growth factor /3 suppresses activation and proliferation of microglia in vitro

Serological, biochemical, and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgG1

T cell receptor Va-V/3 repertoire and cytokine gene expression in active multiple sclerosis lesions

Evidence for effects of B cell stimulatory factor 1 on macrophages: enhancement of the antigen presenting ability of bone marrow-derived macrophages

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (Grant C), the Japanese Ministry of Health and Welfare, the Human Science Foundation, Yamanouchi Pharmaceutical Company Ltd. and Fujita Health University, and in part by the Science Research Promotion Fund from the Japanese Private School Promotion Foundation, Japan. We thank Dr. Hiromi Hosoya for her assistance in flowcytometric analyses.