Interferon gamma up-regulates α2 macroglobulin expression in human astrocytoma cells

Journal of Neuroimmunology

ELSEVIER

Journal of Neuroimmunology53 (1994) 31-37

Interferon gamma up-regulates a2macroglobulin expression in human astrocytoma cells C. Fabrizi

a,

M. Colasanti

a,

T. Persichini

a,

R. Businaro b, G. Starace c, G.M. Lauro a,,

a Dipartimento di Biologia, I l l UniversitY, Viale Ostienze 173, 00154 Rome, Italy b Dipartimento di Scienze Cardiovascolari e Respiratorie, Universit~ "La Sapienza", Rome, Italy c Istituto di Medicina Sperimentale, CNR, Rome, Italy

Received 10 December 1993; revision received and accepted 4 April 1994

Abstract

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor a2macroglobulin (a2M). Interferon gamma (IFNy), a potent immunoregulatorylymphokine,was able to increase the synthesis of aEM by these cells, as measured by ELISA on cell supernatants. The ct2M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures followingIFNy treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFNy. A corresponding enhanced aEM mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of aEM during inflammatory and immune processes in the CNS. An increased release of a2M following IFNy stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes. Key words: a2-macroglobulin; Interferon-y; Astrocytoma; Human I. Introduction

Alpha2-macroglobulin (a2M), one of the major components of plasma, binds to all known proteinases, thereby inhibiting their proteolytic activity (Barett and Starkey, 1973). The formation of complexes aEM-proteinase leads to shifting from the native slow form of the molecule to the fast form which is recognized by a specific cell surface receptor (Sottrup-Jensen, 1989). The OtEM receptor/low density lipoprotein receptorrelated protein (OtEMR) is expressed in several cell types, including macrophages, fibroblasts, astrocytes (Moestrup et al., 1992), and displays different functional roles. Its main action involves the binding with apoliprotein E-enriched lipoproteins (Beisiegel et al., 1989), lipoprotein lipase (Beisiegel et al., 1991), plasminogen activator inhibitor-I complexed with urokinase or tissue-type plasminogen activator (Bu et al., 1992; Nykjaer et al., 1992), besides a2M-proteinase complexes. A receptor-mediated endocytosis mechanism delivers aEM complexes and bound peptides to

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endosomes and lysosomes (Willingham et al., 1980; Nori et al., 1993). The recent knowledge on a2M functions, reviewed by Borth in 1992, claimed its role as a binding and carrier protein for cytokines and growth factors, presumably involved in many important physiological processes, such as development, aging and immune response. In this connection, a recent paper by Chu and Pizzo (1993) reported that proteinase activated a2M can enhance the macrophage antigen processing, by favouring the internalization of antigen via a receptormediated system. It is well known that astrocytes participate in immune responses by presenting antigens to T-lymphocytes in a specific manner, which is restricted by the major histocompatibility complex antigens (MHC) (Fontana et al., 1984); furthermore, interferon gamma (IFNy), a T-cell-derived lymphokine (for review see Farrar and Schreiber, 1993), induces MHC II glycoprotein expression (Wong et al., 1984; Fierz et al., 1985; Colasanti et al., 1993) and cytokine secretion (Sawada et al., 1989) in glial and glioma cells. Our previous results indicated that human astrocytes constitutively synthesize o~2M in the culture (Businaro et al., 1992; Lauro et al., 1992) and express

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C. Fabrizi et al. /Journal of Neuroimmunology 53 (1994) 31-37

a2MR (Nori et al., 1993). If a2M acts as an immunomodulator during several steps of the immune response, its production and its release should be affected by the presence of immunoregulatory substances. In order to verify this hypothesis, we evaluated the expression of a2M in an human astrocytoma cell line (T67) in the presence or absence of IFNy.

reagent (Bio-Rad, Richmond, CA) was added and after 5 min 0D595 was measured versus reagent blank. A standard curve was obtained using different concentration of bovine serum albumin (Sigma, St. Louis, MO) diluted in the same buffer of the samples and plotting absorbance at 595 nm versus protein concentration.

2.4. ELISA 2. Materials and methods

2.1. Cell lines T67 and T70 cell lines (Lauro et al., 1986) were obtained from a III WHO gemistocytic astrocytoma (40th-50th passage in culture) and from a glioblastoma multiforme (40th-45th passage in culture), respectively. The human astrocytoma cells ADF were gifts from Prof. G. Donelli and Dr. W. Malorni, Istituto Superiore di Sanith, Rome, Italy. Fetal human astrocytes were from Prof. F. Gremo, Department of Cytomorphology, University of Cagliari, Italy. All cells were grown routinely in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal calf serum (FCS; SeraLab, Crawley Down, UK) and gentamicin (40 tzg/ml). For all experiments, cells were washed twice with HBSS (Hank's Balanced Salt Solution) and then placed in serum-free medium for 2 days. T67 and T70 cells were characterized as glial fibrillar acidic protein- (GFAP), S-100 protein-, fibronectinand vimentin-positive cells, as previously described (Lauro et al., 1986; Cusimano et al., 1990). ADF glioma cells and fetal astrocytes were also positive for GFAP.

2.2. IFNT treatment 2 × 105 cells were plated in 6-well culture multidishes (Nunc, Denmark) containing DMEM supplemented with 10% FCS and antibiotic. After 2 days the medium was discarded, cells were washed twice with HBSS and then serum-free DMEM with or without human recombinant IFNy (Biogen SA, Geneva, Switzerland; specific activity: 2 x 107 I U / m g protein) was added for different time periods (4, 12, 24, 48, 72 h). At 48 h treatment different doses of IFNy were used: 100, 250, 500, 1000, 2000, 4000 and 6000 U/ml. As assessed by morphological criteria and dye (Trypan blue) exclusion, attached ceils were > 95% viable.

2.3. Determination of proteins After different times of treatment, medium was collected and total amount of proteins in each well was determined by the method of Bradford (1976). Briefly, cellular lysates obtained using 0.1 N NaOH were appropriately diluted and placed in dry test tubes. Dye

Enzyme-linked immunosorbent assay (ELISA) was performed essentially as described by Engvall and Perlmann (1971). Briefly, 96-well ELISA plates (Nunc, Denmark) were incubated with azM purified from human plasma (Sigma, St. Louis, MO) or medium obtained from cultures, both diluted in carbonate buffer (pH 9.7). After incubation overnight at 4°C, plates were coated with 1% BSA in phosphate-buffered saline (PBS), treated first with polyclonal rabbit antibodies directed against a2M, diluted 1:1000 (Dakopatts, Denmark), then with donkey biotinylated anti-rabbit Ig (Amersham, Bucks., UK) and finally with streptavidinbiotinylated horseradish peroxidase complex (Amersham, Bucks., UK). As substrate a solution containing 0.04% o-phenylenediamine (Sigma, St. Louis, MO), 0.1 M citric acid, 0.2 M NazHPO 4 and 0.012% H 2 0 2 was used. The absorbance was read at 492 nm on an automated 310 ELISA reader (Biotech, Cambridge, MA). The unknown concentration in the samples was determined using a standard curve obtained with scalar dilutions of plasma derived azM.

2.5. Polymerase chain reaction (PCR) Total cellular RNA was purified from confluent 5 × 106 T67 cells by the method of Chomczynski and Sacchi (1987). Briefly, a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture was performed. Whole RNA was reverse transcribed into cDNA; the reaction was carried out in a final volume of 20/zl containing 1/zg of total RNA, 50 mM Tris. HCI (pH 8.3), 75 mM KCI, 10 mM dithiothreitol, 3 mM MgC12, 200 ng oligo(dT)12_ls, 200 /xM dNTPs, 10 U RNasin (Promega, Madison, WI), 200 U MMLV (mouse moloney leukemia virus) reverse transcriptase. Before adding RNasin and reverse transcriptase, the reaction mixture was heated to 70°C for 10 min and cooled to 40°C for 5 min and then to room temperature. After 1 h at 37°C the reaction was stopped by heating at 95°C for 10 min and then stored at 4°C until further treatment. Amplification of cDNA was carried out with Thermus aquaticus (Taq) DNA polymerase (Promega, Madison, WI) in a thermal cycler (PerkinElmer Cetus). Samples were prepared by combining 1 /zl of cDNA solution with 49/zl of PCR buffer: 50 mM KC1, 10 mM Tris. HC1 (pH 8.3), 2 mM MgC12, 0.01% (w/v) gelatin, 200 /zM each of the dNTPs, 25 pmol

C. Fabrizi et al. /Journal of Neuroimmunology 53 (1994) 31-37

each of the primers, 2 U Taq DNA polymerase and [a-32p]dCTP (0.125 /zCi/sample; specific activity 400 Ci/mmol; Amersham, Bucks., UK). A MgC12 titration curve had previously established that 2 mM MgC12 was the optimal concentration for this primer pair (data not shown). Samples were overlaid with light mineral oil (Sigma, St. Louis, MO) and heated to 95°C for 3 min. Subsequent cycles were conducted in three temperature steps: 1 min at 94°C, 30 s at 62°C and 1 min at 72°C. After 25 cycles, the samples were incubated at 72°C for an additional 7 min; 10 /zl of each PCR production mixture was electrophoresed in 8% polyacrylamide gel in Tris borate-EDTA buffer. The amplified bands were cut and the radioactivity was measured by /3 counter (LKB, Wallas). Radioactivities in the azM bands were normalized with respect to the/3-actin bands from parallel samples. The primers used were based on known sequence information (Kan et al., 1985) and were located on either side of some introns to ensure that the amplified product was derived from RNA and not contaminating genomic DNA. The sequences for the otzM specific primers are as follows: upstream primer, 5'-GTGCTGTGGACCAAAGCGTG-3' (+1836 to + 1855); downstream primer, 5'-ACAAAGAAGGGCTGGAAGGCT-3' (+ 2424 to + 2404).

2.6. Northern hybridization An aliquot containing 30 /xg of total RNA was applied to a 1% agarose gel containing 10% formaldehyde. After electrophoresis, RNA was transferred by the capillary blot procedure to a nylon membrane (Boehringer Mannheim, Germany). The blots were hybridized with specific probe consisted of a PCR fragment of 584 bp amplified from total RNA of T67 astrocytoma cells. The PCR technique was performed as previously described. The probe was labeled with [a-32p]dATP (specific activity 3000 Ci/mmol; Amersham, Bucks., UK) by random priming reaction. The filters were prehybridized for 2 h at 42°C in 50% formaldehyde, 5 x SSPE, 5 x Denhardt's, 0.1% SDS, 0.1 mg/ml salmon sperm DNA. Hybridization was performed at 42°C overnight with the DNA probe in fresh prehybridization solution. The filters were then exposed to Amersham ECL film with intensifying screens at -70°C. For rehybridization, the membranes were washed in 0.2 mM Tris. HC1 (pH 8), 0.5% SDS solution at 68°C for 1 h. The autoradiography films were evaluated with the Quantimet video programme Leica after recording via camera from the autoradiography film. The Quantimet programme allows the evaluation of the darkness of the selected fields and quantifies them in relative units. The measured darkness of each a2M band was normalized with respect to the

33

correspondent GAPDH band obtained by rehybridization of the same filter.

3. Results T67, an human astrocytoma-derived cell line (Lauro et al., 1986), was shown to produce and secrete a2M in the culture (Businaro et al., 1992). Cells grown in serum-free medium were incubated for 48 h with different doses of IFNy and the accumulation of a2M in the supernatant was measured by an ELISA. IFNy was used at different concentrations (100, 250, 500, 1000, 2000, 4000 and 6000 U/ml) and appeared to stimulate a2M production in a concentration-dependent manner (Fig. 1). The constitutive release of a2M (1.55 ng//xg total protein content) was significantly augmented following a treatment with 250 U / m l of IFN7 with an increment by 1.56-fold; the maximum effect was evident with a dose of 2000 U / m l of IFNy, with an increment by 2.7-fold compared to controls. A plateau was reached as from 2000 U / m l of IFNy. As shown in Fig. 2, when cells were incubated with 2000 U / m l of IFNy for different time periods (4, 12 24, 48 and 72 h), a significant increase in the levels of the released azM could be detected as from 24 h treatment reaching the peak at 72 h. We observed a 2.7-fold constant increment of a2M release in cells treated with 2000 U / m l of IFNy for 24, 48 and 72 h with respect to untreated ceils. Two other additional human glioma cell lines and human fetal astrocytes were also evaluated for baseline and IFNy-stimulated azM expression (Table 1). Cells were treated with 2000 U / m l of IFNy for 48 h. ADF cell line constitutively produced very low levels of

6 $

"~ 5

#

0

_

#

.~3 2 1

0

100

250

5 0 0 1000 2 0 0 0 4 0 0 0 6 0 0 0

Fig. 1. a2Macroglobulin secreted by 2 x 105 T67 cells in serum-free medium following incubation with different doses of IFN,/ for 48 h. For each well the amount of a2macroglobulin was determined by ELISA and the ratio between this value and the total protein content was calculated. Each experiment was carried out in triplicate and statistical analysis was performed using the Bonferroni t-test ( # P < 0.05; * not significant with respect to 2000 U/ml).

C. Fabrizi et al. / Journal of Neuroimmunology 53 (1994) 31-37

34

Table 1 a2Macroglobulin released in cellular supernatants by human astrocytoma (T67 and ADF), human glioblastoma (T70) and fetal astrocytes following 48 h of treatment with IFN~,

10

9

#

O Control

8

• ~rr (2000 u/toO

/

6

~

4

T67

T70

ADF

Fetal astrocytes

Untreated cells 1.55_+0.1 1.68_+0.07 0.16_+0.01 2.25_+0.02 IFNy(2000U/ml) 4.25_+0.34 2.99_+0.15 5.19_+0.02 3.64_+0.2 t-test

P_