Induction of Nitric Oxide Synthase-2 Expression and Measurement of ...

Chapter 17 Induction of Nitric Oxide Synthase-2 Expression and Measurement of Nitric Oxide Production in Enriched Primary Cortical Astrocyte Cultures James A. Hewett and Sandra J. Hewett Abstract Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures. Key words: Astrocytes, Nitric oxide, NO, Nitric oxide synthase-2, Cytokines, Lipopolysaccharide, Interferon-γ, Transforming growth factor-β

1. Introduction Nitric oxide (NO) is a free-radical gas that is produced in biological systems from the amino acid, L-arginine, by the catalytic action of nitric oxide synthase (NOS) (1). NO is a pluripotent endogenous signaling molecule that serves as an important effector in neurotransmission and blood flow (2, 3) and is a key weapon enlisted by the innate immune system in the defense against microbial infection and tumor growth (4). It is also the active metabolite of certain nitrovasodilator drugs, such as nitroglycerin (5). Three unique NOS gene products have been cloned (6–9). NOS-1 and -3 were first identified in neurons and endothelial cells, respectively, and hence were initially termed nNOS and eNOS.

Richard Milner (ed.), Astrocytes: Methods and Protocols, Methods in Molecular Biology, vol. 814, DOI 10.1007/978-1-61779-452-0_17, © Springer Science+Business Media, LLC 2012

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Table 1 Summary of NOS isoform characteristics Isoform

Expression

Activity

Calcium requirement Output

NOS-1

Constitutive

Induced

Dependent

Low

NOS-2

Induced

Constitutive

Independent

High

NOS-3

Constitutive

Induced

Dependent

Low

NO production by these isoforms is activated by elevated intracellular calcium, which triggers NOS catalytic activity via binding of calmodulin (10, 11). NOS-2 was initially cloned from activated macrophages. It is distinguished from the two constitutively expressed, calcium-dependent isoforms in several important respects. First, expression of NOS-2 is not normally detected in cells, but is induced under pathological conditions. Thus, this isoform was initially termed inducible NOS (iNOS). Second, although all NOS isoforms catalyze NO production by a similar enzymatic reaction (10, 11), calmodulin is constitutively bound to NOS-2 and NO production occurs at basal intracellular calcium levels (4). Finally, whereas NOS-1 and -3 catalyze transient bursts of NO production, NOS-2 is persistently active upon expression and hence yields much higher NO output compared to NOS-1 and -3. These properties of the three isoforms are summarized in Table 1. NOS-2 can be expressed by many cell types in addition to those of the macrophage lineage. Within the glial cell population of the central nervous system, for example, astrocytes as well as microglia have the capacity to express NOS-2 (12). Interestingly, oligodendrocytes appear to be incapable of NOS-2 expression (13, 14). With regard to astrocytes, NOS-2 expression and NO production can be induced in vitro by cytokines and/or lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria (15–19). NOS-2 expression has also been reported in astrocytes in vivo following cerebral ischemia and may contribute to the pathogenesis of neuronal injury (20, 21). In this regard, astrocytic NOS-2 activity has been shown to potentiate excitotoxic neuronal injury in cell culture models (22, 23). Astrocytic NOS-2 may also be detrimental in multiple sclerosis (24, 25) and Alzheimer disease (26–28). Thus, there has been much interest in elucidating the mechanisms that control the expression and activity of NOS-2 in astrocytes. The purpose of this chapter is to detail methods to induce and analyze NOS-2 expression in and NO production from astrocyte cultures.

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2. Materials 2.1. General Purpose Solutions

1. Distilled, deionized water (DDW). 2. Phosphate-buffered saline (PBS) 10× stock solution: to make a 10× PBS stock solution, 80 g NaCl, 2 g KCl, 21.6 g Na2HPO4·7H2O, and 2.4 g KH2PO4 are dissolved in 800 mL DDW and the volume is adjusted to 1 L with DDW. Store at room temperature. 3. PBS 1× stock solution: Dilute 10× PBS stock solution to tenfold in DDW (100–900 mL, respectively) and adjust the pH to 7.4.

2.2. Cell Culture

1. Culture media stock (MS): Eagle’s medium containing Earle’s salts, supplemented with 25.7 mM glucose and 28.2 mM sodium bicarbonate. 2. Astrocyte growth medium (GM): MS containing 10% bovine calf serum (Hyclone, Thermo Scientific), 2 mM l-glutamine, 50 IU/mL penicillin, and 50 mg/mL streptomycin (see Note 1) (see Chap. 5 for additional details).

2.3. Cell Stimulation

1. Transforming growth factor-β1 (TGF-β1) stock solution (4 μg/mL): recombinant human TGF-β1 (R&D Sys) is reconstituted in DDW containing 1 mg/mL fatty acid-free bovine serum albumin and 4 mM hydrochloric acid, sterilized by filtration, and stored in small aliquots at −80°C. 2. Interleukin-1β (IL-1β) stock solution (10 μg/mL): recombinant mouse IL-1β (R&D Sys) is reconstituted in PBS containing 0.1% fatty acid-free bovine serum albumin, sterilized by filtration, and stored in small aliquots at −20°C in a manual defrost freezer (see Note 2). 3. Interferon-γ (IFN-γ) stock solution (50 μg/mL): recombinant mouse IFN-γ (R&D Sys) is reconstituted in sterile PBS containing 0.1% fatty acid-free bovine serum albumin, aliquoted, and stored at −80°C (see Note 3). 4. LPS stock solution (2 mg/mL): E. coli (0127:B8) LPS is reconstituted in DDW, filter-sterilized, and stored in 1 mL aliquots at −20°C (see Note 4).

2.4. Griess Assay Reagents

1. Solution 1: 1.32 g sulfanilamide is added to 39 mL of DDW, and 60 mL glacial acetic acid is slowly added with gentle mixing until solid is completely dissolved. Store at 4°C. 2. Solution 2: 0.1 g N-(1-naphthyl)ethylenediamine dihydrochloride is added to 100 mL DDW. Store protected from light (e.g., in a brown plastic bottle) at 4°C.

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3. Nitrite (NO2−) stock solution: to make a 1 M stock solution of the NO2− standard, 0.69 g sodium NO2− is dissolved in 10 mL DDW and stored tightly capped at 4°C. 2.5. Immunocytochemistry

1. Paraformaldehyde fixative solution (4%): add 4 g paraformaldehyde to 89 mL DDW, loosely cap, and stir on a hotplate in a fume hood to ~60°C. Add 10 N NaOH drop-wise until the solution clears. After cooling, add 10 mL 10× PBS and adjust the pH to 7.4. Store tightly capped at 4°C for several days (see Note 5). 2. Triton X-100 stock solution (10%): add 10 mL Triton X-100 to 90 mL DDW. 3. Sodium azide stock solution (10%): dissolve 1 g sodium azide in 9 mL DDW and adjust the volume to 10 mL (see Note 6). 4. Normal goat serum (Jackson ImmunoResearch): Reconstitute the lyophilized powder in 10 mL DDW, and store in aliquots of 1 mL at −20°C. 5. Blocking/permeabilization solution: 5% normal goat serum, 0.25% triton X-100, and 0.05% sodium azide in PBS (see Note 7). 6. Antibody dilution buffer: 2% normal goat serum, 0.05% sodium azide in PBS. 7. Anti-Nitric Oxide Synthase-2 (NOS-2) primary antibody: store rabbit anti-NOS-2 antibody (0.2 mg/0.2 mL, Millipore) in 25 μL aliquots at −20°C. 8. Anti-glial fibrillary acidic protein (GFAP) primary antibody: store rat anti-GFAP antibody (0.1 mg/0.2 mL, Invitrogen) in 25 μL aliquots at −20°C. 9. Secondary antibody for anti-NOS-2 antibody: reconstitute Cy3-conjugated goat anti-rabbit antibody (1.5 mg/mL, Jackson ImmunoResearch) in 1.1 mL DDW and store protected from light in 75 μL aliquots at −20°C. 10. Secondary antibody for anti-GFAP antibody: reconstitute FITC-conjugated goat anti-rat antibody (1.4 mg/mL, Jackson ImmunoResearch) in 0.4 mL DDW and store protected from light in 25 μL aliquots at −20°C. 11. DAPI nucleic acid staining reagent: dilute 10 mg DAPI (dilactate form, Molecular Probes) in 2 mL DDW and store protected from light in aliquots of 50 μL at −20°C. Immediately prior to use, dilute 1/2,500 in PBS. 12. Prepare primary antibody solution immediately prior to use by diluting rat anti-GFAP and rabbit anti-NOS-2 antibodies to 5 and 2 μg/mL (1/100 and 1/500), respectively, in antibody dilution buffer.

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13. Prepare the secondary antibody solution immediately prior to use by diluting goat anti-rat FITC-conjugated and goat antirabbit Cy3-conjugated antibodies (15 and 7.5 μg/mL, respectively) in antibody dilution buffer (see Note 8).

3. Methods 3.1. Culturing Highly Enriched Cortical Astrocyte Monolayers

1. Harvest brains from postnatal day 1 mice, dissect cortices, and digest with trypsin. 2. Pellet single cell suspensions by centrifugation, resuspend in GM, and plate on 24-well Falcon Primaria tissue culture plates (BD Biosciences) at a density of two cerebral hemispheres/ plate in a volume of 0.4 mL/well. 3. Incubate the plates at 37°C in a humidified atmosphere of 5% CO2/95% air until monolayers of astrocytes are established. 4. Treat the monolayers for 5–7 days with 8 μM cytocine arabinoside (AraC) to selectively prevent microglia proliferation without affecting contact-inhibited astrocytes. 5. One to 2 days prior to stimulation, treat the monolayers with 50–75 mM leucine methyl ester (LME) to eliminate residual contaminating microglia (see Note 9).

3.2. Cell Stimulation and Harvest of Supernatants for Analysis of NO Production

1. Stock reagents of stimuli are diluted in GM to 40× final concentration (TGF-β1 = 0.12 μg/mL; IL-1β = 0.04 μg/mL; IFN-γ = 0.12 μg/mL; LPS = 40 μg/mL) (see Note 10). 2. Add 0.01 mL of diluted stimuli or GM to wells containing 0.4 mL GM, gently mix the plates by swirling, and return to the 37°C incubator. 3. After incubating for 24 h in the presence of stimuli, transfer duplicate 0.1 mL samples of cell supernatant to 96-well plates, cover with a sheet of parafilm, and store at −20°C in a manual defrost freezer.

3.3. Analysis of NOS-2 Protein Expression by Immunocytochemistry

1. Aspirate the remaining stimulation media from wells using a vacuum and wash the cell monolayers once with 0.4 mL/well cold PBS 2. Aspirate the wash solution and add 0.4 mL 4% paraformaldehyde fixative solution to each well. 3. After incubating cells for 30 min at room temperature in the fixation solution, wash the wells twice with 0.4 mL cold PBS and add 0.3 mL of blocking/permeabilization solution to each well. 4. Incubate the monolayers overnight at 4°C (see Note 11).

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5. Aspirate the blocking solution, add 0.2 mL primary antibody solution, and incubate overnight at 4°C with gentle rocking. Incubate two wells without primary antibodies to assess the nonspecific binding of secondary antibodies (see Note 11). 6. Aspirate the primary antibody solution and add 0.4 mL/well PBS at room temperature. After 5 min, aspirate the wash and repeat for a total of three washes. 7. Aspirate the PBS from the wells, add 0.2 mL secondary antibody solution, and incubate at room temperature, protected from light for 1–2 h. 8. Wash wells as in step 6. 9. Aspirate the PBS from wells, add 0.2 mL DAPI solution diluted 1/2,500 in PBS, and incubate at room temperature protected from light for 0.5–1 h. Alternatively, DAPI can be added to the secondary antibody solution (step 7). 10. Wash the wells as in step 6, add 0.4 mL PBS and acquire fluorescent images using a microscope outfitted for epifluorescence analysis and digital image acquisition. Images can be modified in Adobe Photoshop (see Notes 12 and 13). 11. Typical results of NOS-2 expression in astrocyte cultures are shown in Fig. 1. 3.4. Analysis of NO Production by the Griess Assay for Nitrite

Nitrite (NO2−) is a major stable metabolite of NO in aqueous solutions (29). It can be quantified in biological solutions via the Griess assay (30), which has been employed extensively as an indirect measure of NOS-2 activity. The Griess assay is a simple colorimetric assay, whereby NO2− in cell supernatants reacts with sulfanilamide in acidic solution and the product is coupled with N-(1-naphthyl) ethylenediamine dihydrochloride (31, 32). The resulting deep red azo dye is quantified spectrophotometrically at 550 nm using a standard curve generated with sodium NO2−. 1. Prepare the color reagent immediately prior to the assay by mixing equal parts Griess solutions 1 and 2, avoiding crosscontamination of stock solutions. 2. Dilute the NO2− stock solution to 100 μM in GM (see Note 14). 3. Prepare a NO2− standard curve for each 96-well plate of samples to be assayed by serial dilution as follows: (a) 100 μM NO2−: add 0.1 mL of 100 μM NO2− solution in duplicate wells. (b) 50 μM NO2−: add 0.1 mL of 100 μM NO2− solution to 0.1 mL of GM in duplicate wells and mix thoroughly by trituration. (c) 25 μM NO2−: transfer 0.1 mL from wells containing 50 μM NO2− to duplicate wells containing 0.1 mL GM and mix thoroughly by trituration.

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Fig. 1. Analysis of NOS-2 protein expression in astrocytes. Cultures were stimulated as described in Subheading 3.2 and NOS-2 protein expression was assessed by immunocytochemistry as described in Subheading 3.3. (a–c) Astrocyte cultures treated in the absence of any stimulus. (d–f) Astrocyte cultures treated with TGF-β1, IFN-γ, and LPS. Images in each treatment group were from the same microscopic field. (a, d) GFAP expression; (b, e) NOS-2 expression; (c, f) DAPI nuclear stain.

(d) 12.5 μM NO2−: transfer 0.1 mL from wells containing 25 μM NO2− to duplicate wells containing 0.1 mL GM and mix thoroughly by trituration. (e) 6.25 μM NO2−: transfer 0.1 mL from wells containing 12.5 μM NO2− to duplicate wells containing 0.1 mL GM and mix thoroughly by trituration. (f) 3.12 μM NO2−: transfer 0.1 mL from wells containing 6.25 μM NO2− to duplicate wells containing 0.1 mL GM and mix thoroughly by trituration.

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Fig. 2. Representative Griess assay standard curve. Sodium nitrite was serially diluted in GM on a 96-well plate and assayed as described in Subheading 3.4.

Fig. 3. Analysis of NO production from astrocytes. Cultures were pretreated without (−) or with (+) TGF-β1 for 16–18 h and stimulated with IL-1β (I), LPS (L), and/or IFN-γ (G) as described in Subheading 3.2. 0 = no stimuli (GM only). Nitric oxide (NO) production (i.e., NOS-2 activity) was quantified 24 h after I,L, G treatment by the Griess assay for nitrite (see Subheading 3.4), a stable metabolite of NO.

(g) 1.56 μM NO2−: transfer 0.1 mL from wells containing 3.12 μM NO2− to duplicate wells containing 0.1 mL GM and mix thoroughly by trituration. (h) Blank: add 0.1 mL GM to duplicate wells. (i) A representative standard curve is shown in Fig. 2. 4. Add 0.1 mL color reagent to each well of cell supernatant and NO2− standard and incubate at room temperature for approximately 10 min. 5. Measure NO2− levels at 550 nm using a computerized plate reader spectrophotometer and software (e.g., SpectraMax M2, Molecular Devices) (see Note 14). 6. Results from a typical experiment are shown in Fig. 3 (see Note 15).

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4. Notes 1. Calf serum and other culture reagents with the lowest endotoxin level available (typically