Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor

"' J o u r n a l of Neuroimmunology ELSEVIER

Journal of Neuroimmunology 50 (1994) 101-107

Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor Barbara P. Barna a,,, James Pettay b, Gene H. Barnett b, Ping Zhou b, Koichi Iwasaki b, Melinda L. Estes b a Department of Clinical Pathology, L-I2, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-5131, USA b Departments of Neurosurgery, Neurology, and Anatomic Pathology, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA (Received 24 August 1993) (Revision received 22 October 1993) (Accepted 22 October 1993)

Abstract

Infiltration of the central nervous system (CNS) by monocytes is a characteristic of many non-malignant disease processes, although the signals regulating such traffic are unclear. Tumor necrosis factor (TNF) and other inflammatory cytokines have been shown to elicit production of monocyte chemoattractant activity in glioma cells, but the regulation of such activity in non-neoplastic adult astrocytes has not been examined. We previously observed that TNF constituted a proliferative signal for non-neoplastic adult human astrocytes in vitro involving the 55-kDa TNF receptor. In the present study, we demonstrate that TNF exposure enhances the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and functional monocyte chemoattractant activity in non-neoplastic astrocytes. Results indicated that MCP-1 mRNA expression was maximal within 3 h, and was further augmented by the protein synthesis inhibitor cycloheximide (CY). Antibody (htr-9) directed against the 55-kDa TNF receptor also elicited MCP-1 mRNA expression while antibody to the 75-kDa TNF receptor (utr-1) was ineffective. Secretion of monocyte chemoattractant activity was significantly greater in TNF- or htr-9-treated astrocytes than in utr-l-treated or untreated controls; activity was abolished by treatment with antibody to MCP-1. These findings suggest that non-neoplastic adult human astrocytes may contribute to CNS inflammatory responses by mediating recruitment of peripheral blood monocytes. Key words: Astrocyte; Tumor necrosis factor; Monocyte chemoattractant protein-l; Astrocytoma

1. Introduction The production of factors eliciting monocyte chemotaxis is a well-recognized property of many types of malignant human cells, including astrocytoma (Graves et al., 1989; Matsushima et al., 1989; Yoshimura et al., 1989a; Graves and Valente, 1991), and one which has contributed to the identification of a novel family of pro-inflammatory proteins. Monocyte chemotactic protein-1 (MCP-1) is an example of this chemokine family which is characterized by the presence of two adjacent cysteine residues, and the preferential attraction of

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monocytes but not neutrophils (Furutani et al., 1989; Yoshimura et al., 1989b; Rollins, 1992; Van Damme et al., 1992). MCP-1 bears a high degree of homology with the murine JE gene, which was originally identified in mitogen-stimulated non-neoplastic fibroblasts (Rollins et al., 1988). Non-neoplastic human somatic cells such as fibroblasts (Strieter et al., 1989; Yoshimura and Leonard, 1990), endothelial cells (Strieter et al., 1989; Rollins et al., 1990), and mesangial cells (Rovin et al., 1992) are also capable of MCP-1 production, which is significantly augmented by exposure to tumor necrosis factor (TNF) or interleukin-1 (IL-1). Monocytes, the primary source of such cytokines, similarly respond to T N F with increased MCP-1 production in an autocrine manner (Rollins et al., 1989; Colotta et al., 1992).

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B.P. Barna et al. /Journal of Neuroimmunology 50 (1994) 101-107

MCP-1, in addition to monocyte chemoattractant properties, directly upregulates monocyte cytokine production (Jiang et al., 1992) and cytostatic activity (Matsushima et al., 1989). Evidence for production of MCP-1 within the central nervous system (CNS) during inflammatory conditions, has been derived from two different studies of experimental autoimmune encephalomyelitis EAE (Hulkower et al., 1993; Ransohoff et al., 1993). In the latter model, expression of JE/MCP-1 mRNA was associated with astrocytes as demonstrated by the presence of the astrocytic marker, glial fibrillary acidic protein (GFAP) in JE/MCP-l-reactive ceils (Ransohoff et al., 1993). In human disease, monocytic infiltrates are especially prominent in multiple sclerosis (Traugott et al., 1984; Estes et al., 1990) and in post-infectious encephalitides (Ambler et al., 1971; Hickey, 1991). Examination of the CNS in both humans and experimental animals has indicated the presence of TNF during such conditions (Grau et al., 1987; Leist et al., 1988; Hofman et al., 1989; Selmaj et al., 1991; Merrill et al., 1992). The major cellular contributors of TNF may be microglia (Sawada et al., 1989; Lee et al., 1993); however, astrocytes have also been shown to be capable of TNF production (Lieberman et al., 1989; Chung and Benveniste, 1990). Collectively, these obser: vations suggest that the localized production of MCP-1 in response to TNF or other inflammatory cytokines may represent an amplification mechanism for the sustained recruitment and activation of mononuclear phagoc~es. The relationship between TNF exposure and the participation of non-neoplastic astrocytes in monocyte recruitment, however, has not been examined. The purpose of the present study was to characterize the effects of TNF on regulation of MCP-1 production in non-neoplastic adult human astrocytes. Our results indicated that brief exposure to TNF rapidly elevated non-neoplastic astrocyte MCP-1 mRNA expression and subsequent secretion of biologically active monocyte chemoattraetant, This ehemoattractant activity was neutralizable by antibody to MCP-1. Further, the effect of TNF on astrocyte MCP-1 was specifically related to stimulation of the 55-kDa TNF receptor. Involvement of this receptor in MCP-1 production, to our knowledge, has not been described previously.

al., 1990; Barna et al., 1993). The non-neoplastic astrocytic cell lines, PIN and W3N, have also been described (Barna et al., 1990; Estes et al., 1990), and were established from adult tissue removed for treatment of epilepsy. Early passages of cells were preserved in cryogenic storage. Astrocytoma ceils were studied at passages 20-40, and non-neoplastic astrocytes at passages 2-4. By immunocytochemical staining, the astrocytic marker, glial fibrillary acidic protein (GFAP), had been detected in CCF-S'ITG1 ( > 70%), WITG3 (20%), and in over 90% of P1N and W3N. All cells were free of mycoplasma or other microbial contamination. Growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin (Gibco, Grand Island, NY). Media were routinely monitored for endotoxin contamination by Limulus amoebocyte lysate assay (Woods Hole, MA), and levels were less than 0.02 ng/ml. 2.2. Cell stimuli Recombinant human TNF was obtained from Genzyme Corporation (Cambridge, MA) (specific activity, 20 units/ng). Murine monoclonal antibodies utr-1, directed, against the 75-kDa TNF receptor, and htr-9, directed against the 55-kDa TNF receptor (Brockhaus et al., 1990; Espevik et al., 1990) were generous gifts from Dr. Manfred Brockhaus, Hoffman-La Roche, Basel, Switzerland. 2.3. RNA extraction and analysis

RNA was isolated from non-neoplastic and neoplasiic astrocyte monolayers cultured as described above. The single-step acid guanidium thiocyanate-phenolchloroform extraction method of Chomczynski and Sacchi (1987) was employed, using RNAzol (Cinna/ Biotecx, Friendsville, TX). The R N A pellet was dissolved in DEPC (diethylpyrocarbonate)-treated water and quantified by UV absorbance at 260 nm. 2. 4. DNA probes

2. Materials. and methods

DNA probes for JE/MCP-1 (American Type Culture Collection), or/3-aetin (gift from T. Nielsen) were labelled with 20 t~Ci [a-32P]dCTP (Amersham, Arlington, IL; specific activity 300 Ci/mmol) per probe using a nick translation kit (Boehringer Mannheim, Indianapolis, IN) as described by the manufacturer.

2.1. Astrocyte culture

2.5. RNA analysis

The human: astrocytoma cell lines, CCF-STTG1 and WITG3, were. established from biopsy specimens obtained during surgery for tumor diagnosis, and have been described in detail (Estes et al., 1990; Milsted et

For Northern blot analysis, total RNA (20 /zg) in 60% formamide, 1 × 4-morpholinepropanesulfonic acid (MOPS) buffer and 11% formaldehyde was heat-denatured (65°C, 15 min), quenched on ice, then size-frac-

B.P. Barna et al. /Journal of Neuroimmunology 50 (1994) 101-107

tionated by electrophoresis in a 1.0% agarose gel containing 2.2 M formaldehyde for 3 h at 100 V as previously described (Barna et al., 1993). Following electrophoresis, ethidium bromide stain was used to identify RNA molecular mass standards or 28S and 18S RNA run in control lanes. RNA was then transferred overnight to a GeneScreen membrane (Dupont, Boston, MA) with 25 mM sodium phosphate (pH 6.5). After transfer, the membrane was baked under vacuum at 80°C for 2 h and prehybridized in 50% formamide, 2 × 1,4-piperazine-dithane-sulfonic acid (PIPES), 0.5% sodium dodecyl sulfate (SDS), poly(A) (10 mg/ml) and denatured salmon sperm DNA (5 mg/ml) at 42°C for 4 h. Hybridization was carried out at 42°C for 18 h in prehybridization solution containing 106 cpm/ml of heat-denatured, nick-translated eDNA probe. The membrane was washed in 2 × SSC + 0.1% SDS once at room temperature (30 min), once at '65°C (30 min), and twice at 65°C (15 min). For autoradiography, the membrane was exposed to Kodak XAR-5 film with intensifying screens for 24-48 h at -70°C. The membrane was then stripped and rehybridized with the remaining probe. For slot blot analysis, total RNA (5-10 /zg), after heat-denaturing and quenching, was brought to a final Sample volume of 200 /zl by addition of 10 x standard saline citrate (SSC). Samples were applied to a GeneScreen membrane with gentle vacuum, washed twice with 10 × SSC, and the membrane was air-dried, baked, prehybridized, and hybridized as described above. Enhancement of JE/MCP-1 gene expression was determined as a qualitative increase in comparison to gene expression in non-TNF-treated cells. Expression of/3-actin mRNA was evaluated as an internal control to normalize the amount of RNA applied from each specimen.

103

2.6. Preparation of astrocyte-conditioned media (CM) Confluent monolayers of astrocytes in T-25 culture flasks were washed in serum free RPMI 1640 medium, and incubated for 6 h in serum-free RPMI 1640 alone or medium containing 10 n g / m l recombinant TNF, or 10 /zg/ml monoclonal antibody utr-1 or htr-9. After media were discarded, flasks were washed again, and reincubated with fresh serum-free medium without TNF or other stimuli for an additional 42 h. At the end of incubation, conditioned media were removed, centrifuged, and stored at -70°C in aliquots for evaluation of chemoattractant bioactivity.

2. 7. Monocyte chemotaxis assay Human peripheral blood mononuclear leukocytes (MNL) were obtained by Ficoll-Hypaque (Pharmacia LKB, Pisacataway, N J) centrifugation of heparinized blood from normal donors. Chemotaxis was carried out in Boyden Chambers as described by Snyderman et al. (1972). 100/.d of MNL suspension (5 × 10 -s cells/ml in RPMI 1640 containing 2% bovine serum albumin + 1% HEPES buffer), were added to the upper portion of a blind-well chamber containing a 5-/zm porosity polycarbonate membrane (Neuro Probe, Cabin John, MD) and one of the following solutions in the bottom portioni 1 × lO-S: M n-formyl-methionyl-phenylalanine (nfmp) (Sigma, St. Louis, MO) (positive control); astrocyte-conditioned media; or control media (negative control). Chambers were incubated for 90 min at 37°C; membranes were removed, rinsed, stained with Wright-Giemsa, and mounted on slides. Non-specific esterase staining (Sigma, St. Louis, MO) was used to determine the percentages of monocytes in migrating

~ACTIN

ASTROCYTE CULTURES TREATMENT

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Fig. I. Time-dependent expression of JE/MCP-I m R N A in astrocytes exposed to culture medium with or without T N F a (10 n g / m D for 1.5~24 h. (a) Non-neoplastic astrocyticcell lines PIN and W3N; (b) glioblastoma WITG3. Figures depict JE/MCP-I and as a control for R N A loading, fl-actJn m R N A .

B.P. Barna et aL /Journal of Neuroimmunology 50 (1994) 101-107

104

populations. The numbers of monocytes migrating through each membrane were counted microscopically using a grided eye piece and 400 x magnification. Results were expressed as mean numbers of monocytes/10 high power fields (HPF)+_ SD (n = 3 replicate chambers/variable). For comparative studies, results with astrocyte-conditioned media were also expressed as a percentage of nfmp-elicited chemotaxis (chemotactic activity) as described by Barker et al. (1991).

2.8. Immunoadsorption of MCP-1 by antibody Specific murine antibody to human MCP-1 was a generous gift from Dr. Teizo Yoshimura, National Cancer Institute, Frederick, MD. Protein A-Sepharose beads (Pierce Chemical, Rockford, IL) (35 /zl) were mixed with 100 ~g anti-MCP-1 or normal murine IgG1 for 1 h at 4°C, centrifuged, washed and rinsed with RPMI 1640 medium containing 1% human AB serum. Astrocyte conditioned media (50/~1) were mixed with coated beads for 1 h at 4°C and centrifuged. Supernatant fluids were removed and assayed for chemoattractant activity.

2.9. Biostatistical analysis The statistical significance of differences in monocyte migration toward putative stimuli vs. medium controis were analyzed by Student's t-test. All experiments were carried out in duplicate unless otherwise noted.

3. Results

3.1. Regulation of MCP-I mRNA expression Examination of two human non-neoplastic astrocytic lines, P1N and W3N, indicated that TNF augmented W3N

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Fig. 2. Effect of cyclobe~nide (CY) on JE/MCP-1 mRNA expression of astrocytes exposed for 3 h to culture medium with or without TNFa (10 ng/ml), CY (1/zg/ml), or a combination thereof, mRNA was derived from a non-neoplastic astrocytic cell line (W3N) and two glioblastoma lines (WITG3 and STTG1).

MCP-1 mRNA in a time-dependent manner, with maximal expression detectable at 1.5-3 h (Fig. 1A). As a positive control, we utilized a glioblastoma cell line (WITG3), since neoplastic astrocytic cell lines have been reported to express MCP-1 constitutively and in response to TNF (Kasahara et al., 1991). Results confirmed that TNF augmented expression of a 0.8-kb MCP-1 mRNA transcript in WITG3 cells in a similar time course with maximal expression at 3 h (Fig. 1B). The size of transcribed MCP-1 mRNA was comparable to that reported elsewhere for human cells (Rollins et al., 1989). To determine whether non-neoplastic astrocyte expression of MCP-1 mRNA was sensitive to regulation requiring protein synthesis, non-neoplastic astrocytes together with neoplastic astrocytoma cells (as controls) were exposed to TNF for 3 h in the presence and absence of CY. Expresston of JE/MCP-1 mRNA in response to TNF was not inhibited by CY but was augmented further (Fig. 2). These findings demonstrated that expression of JE/MCP-1 mRNA was post-transcriptionally regulated by an inhibitory pro-

WITG3

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Fig. 3. Effects of TNF receptor-binding monoclonal antibodies, utr-1 and htr-9 (10 ~g/ml), on non-neoplastic astrocyte (W3N) and glioblastoma (WITG3) expression of JE/MCP-1 mRNA and secretion of monocyte chemoattractant activity into conditioned media (CM). Astrocytes were exposed to TNFa (10 ng/ml), utr-1, htr-9, or medium alone for 3 h before extraction of total cellular mRNA. To prepare CM, astrocytes were first exposed to reagents shown for 6 h, and reeultured in medium alone for 42 h. (a) MRNA expression; (b) monocyte chemoattractant activity in CM. Data represent mean percentage monocyte chemotactic activity (:1: SD) relative to that elicited by nfmp (n = 3). * P < 0.01 compared to Medium.

B.P. Barna et al. /Journal of Neuroimmunology 50 (1994) 101-107

tein in non-neoplastic as well as neoplastic human astrocytes. Examination of TNF receptor involvement indicated that treatment with monoclonal antibody (htr-9) directed against the 55-kDa TNF receptor was comparable to TNF with respect to enhancement of MCP-1 mRNA expression in non-neoplastic astrocytes and in astrocytoma cells (Fig. 3A). In contrast, monoclonal antibody (utr-1) directed against the 75-kDa TNF receptor was not effective in either cell type (Fig. 3A).

80 v//.,,]

In order to determine whether JE/MCP-1 mRNA expression led to secretion of biologically active protein product, we examined astrocyte-conditioned media for evidence of monocyte chemoattractant. Astrocytes were incubated with TNF or monoclonal antibodies to TNF receptors for 6 h, extensively washed, and reincubated for 42 h in TNF-free medium. Chemotaxis assays were carried out to examine the motility of normal human mononuclear leukocytes in response to conditioned media removed from astrocyte cultures. Results indicated that TNF-treated non-neoplastic and neoplastic astrocytes secreted significantly higher titers of chemoattractant activity into conditioned media than did untreated astrocytes (Fig. 3B). Non-specific esterase cytochemistry characterized migrating mononuclear leukocytes as > 99% monocytes (data not shown). Treatment of neoplastic and non-neoplastic astrocytes with monoclonal antibodies to TNF receptors

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105

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Fig. 5. Effect of antibody to MCP-1 on monocyte chemoattractant activity of conditioned media (CM) from non-neoplastic astrocytes (W3N) or glioblastoma (WITG3) cells exposed to TNFa (10 ng/ml) as described in the legend to Fig. 3. CM were treated with anti-MCP-1 antibody conjugated to Protein A-Sepharose 4B (AB/MCP-1) or normal IgG/Protein A conjugate (Ig-NORM), and tested for chemoattractant activity at a l / 5 dilution. Data represent mean numbers of monocytes/10 high power fields (n = 3); * P < 0.005 compared to Untreated.

yielded evidence of a selective effect on monocyte chemoattractant secretion (Fig. 3B). Only antibody htr9 directed against the 55-kDa TNF receptor provoked chemoattractant activity, while antibody utr-1 to the 75-kDa receptor was ineffective (Fig. 3B). These results complemented those of mRNA studies (Fig. 3A). Analysis of monocyte chemoattractant activity present in dilutions of conditioned media from TNFtreated astrocytes indicated that the effect was dosedependent (Fig. 4). To confirm the involvement of MCP-1 in astrocyte-secreted monocyte chemoattractant activity, astrocyte-conditioned media were exposed to activated Sepharose 4B/Protein A-conjugates bound with either antibody to human MCP-1 or normal murine IgG1. Treatment with anti-MCP-1 conjugate significantly reduced monocyte chemoattractant activity in conditioned media from both non-neoplastic astrocytes and glioblastoma ceils, whereas treatment with normal IgG conjugate had no effect (Fig. 5).

.

4. D i s c u s s i o n

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DILUTION OF CONDITIONED M E D I A Fig. 4. Dose-dependence o f monocyte chemoattractant activity in

42-h conditioned media (CM) from non-neoplastic astrocytes (W3N) or glioblastoma cells (WITG3). Astrocytes were exposed to TNFa (10 ng/ml) or medium alone and CM derived as described in the legend to Fig. 3. Data represent mean percentage monocyte chemotactic activity ( -1-SD) (n = 3) relative to chemotaxis elicited by nfmp. No CM (square); * P < 0.05; * P < 0.001 compared to CM from astrocytes exposed to medium alone.

The regulation of MCP-1 expression has been shown to be dependent upon cell lineage. Colotta and co-investigators (1992) demonstrated that the regulation of MCP-1 gene expression in mononuclear phagocytes differed from that of non-hematopoietic cells such as endothelial or smooth muscle cells. In human monocytes, treatment with CY blocked MCP-1 mRNA expression elicited by TNF, IL-1, or LPS (Colotta et al., 1992). In contrast, CY superinduced MCP-1 mRNA expression in smooth muscle, endothelial, and fibrosarcoma cells (Colotta et al., 1992). Our observation that CY did not diminish MCP-1 mRNA expression in

106

B.P. Barna et al. /Journal of Neuroimmunology 50 (1994) 101-107

non-neoplastic human astrocytes suggests that these cells resemble other non-hematopoietic cell types in that respect. Also, the rapid elevation of MCP-1 mRNA in response to TNF is in keeping with other studies of TNF-treated astrocytoma cells (Kasahara et al., 1991). Many characteristics of astrocytes resemble those of keratinocytes, most probably because of a common ectodermal origin from neural crest. Both cell types have been found to display similar tissue-specific regulation of major histocompatibility complex antigen expression in response to interferon gamma (IFNy) treatment (Massa and ter Meulen, 1988). IFN3, also stimulates keratinocyte production of monocyte chemotactic and activating factor (MCAF), now known to be identical to MCP-1 (Matsushima et al., 1989; Yoshimura et al., 1989b). TNF, however, does not stimulate this activity in keratinocytes (Barker et al., 1991), whereas our findings indicate stimulation in astrocytes. Thus, with respect to production of monocyte chemoattractant factors, astrocytes are sensitive to TNF exposure, whereas keratinocytes appear to be refractory. In previous studies, we observed that TNF augmented proliferation of non-neoplastic human astrocytes, but inhibited that of glioblastoma cell lines (WITG3 and ST-I'G1) (Barna et al., 1993). Similarly, antibody to the 55-kDa TNF receptor mimicked these disparate effects of TNF on neoplastic and non-neoplastic astrocyte proliferation (Barna et al., 1993). In contrast, the current data indicate that both neoplastic and non-neoplastic astrocytes respond comparably to TNF or antibody to the 55-kDa TNF receptor in terms of MCP-1 production. Results suggest that the effects of TNF on MCP-1 production may be independent of the effects on proliferation, and that at least two different mechanisms distal to the TNF receptor are involved. These findings also emphasize the critical involvement of the 55-kDa TNF receptor in transduction of TNF signalling in astrocytes. In summary, these data further illustrate the regulatory effects of TNF on non-neoplastic human astrocytes via the 55-kDa TNF receptor, specifically the production of MCP-1. The possibility of additional monocyte chemoattractants elicited by TNF is not ruled out, however. The evidence for localized TNF production within the CNS indicates that astrocytes may be exposed to this cytokine via paracrine or autocrine pathways. Our studies suggest that the consequences of relatively brief exposure to TNF may be augmentation of astrocyte MCP-1 production and subsequent amplification of monocyte recruitment to the CNS.

Acknowledgements The authors wish to thank the members of the Department of Neurosurgery for their assistance in

providing surgical material, Ms. Barbara Jacobs and Ms. Joyce Antal for technical assistance, and Dr. Manfred Brockhaus for critical review of the manuscript. This work was supported in part by Grants NIH-1454HL (M.L.E), NCI-R01 CA49950 (B.P.B.), and by a grant from the Epilepsy Foundation of America (M.L.E., B.P.B.).

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