Chemiluminescence and nitrite determinations by the MALU macrophage cell line

JOURNALOF IMMUNOLOGICAL METHODS ELSEVIER

Journal of ImmunologicalMethods 174 (1994) 259-268

Chemiluminescence and nitrite determinations by the MALU macrophage cell line F. B o u d a r d

*, N . V a l l o t , C. C a b a n e r ,

M. Bastide

Laboratoire d'Immunologie et Parasitologie, Facult~ de Pharmacie, Avenue Charles Flahault, 34060 Montpellier Cedex 1, France

Abstract

The MALU macrophage cell line is an in vitro coculture of mesothelial cells and alveolar macrophages from mice. During the culture, macrophages in a preactivated state can be collected in the supernatant (Lombard et al., 1988). We describe here two methods to measure reactive oxygen intermediate (ROI) and reactive nitrogen intermediate (RNI) production by the MALU macrophages. We measured ROI by the chemiluminescence assay with a luminometer and RNI was measured colorimetrically in a spectrophotometer with the Griess reagent. Different parameters (cell number, incubation time, temperature and activating substance) which can interfere with the cell response were analysed. Our results show that the MALU macrophage cell line produces large amounts of ROI and RNI after activation. This cell line is a good model for investigating the effect of pharmacological drugs on ROI and RNI production. Keywords: Macrophage; Reactive oxygen intermediate; Reactive nitrogen intermediate; Chemiluminescence; Griess

reagent

1. Introduction

L o m b a r d et al. (1988) described an in vitro coculture of mesothelial cells and alveolar macrophages from lung explants of C57B1/6 L p r / L p r mice ( M A L U macrophages). U n d e r appropriate conditions, this coculture can be infinitely reseeded and macrophages can proliferate; they are released in the fluid phase and can be collected in the supernatant. These macrophages have b e e n characterized by the following criteria: their mor-

* Corresponding author.

phology and adherence properties, their phagocytic activity, the presence of functional Fc and mannose receptors and m e m b r a n e ectoenzymes. These cells generate reactive oxygen radicals and are cytotoxic for tumor cells. During the coculture, growth and activation factors, such as MCSF (CSF-1) and IFN-3,, are released in the medium. Phenotypic studies have shown that these ceils are in a high differentiation state: they express the m a c r o p h a g e markers (Mac-l, Mac-2 and F 4 / 8 0 ) and M H C class I antigens; they do not express the markers of T and B cells (Lombard et al, 1988; Falkenberg et al., 1992). Macrophages play an important role in the

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F. Boudard et al. /Journal of lmmunological Methods 174 (1994) 259-268

immune response because they are involved in host defence mechanisms toward infections and tumors (Neveu, 1986). When cells are stimulated with a particular substance (opsonised zymosan) or a protein kinase C activating substance (phorbol-myristate-acetate, PMA), a marked increase in oxygen consumption associated with glucose oxidation via the hexose monophosphate shunt is observed. This phenomenon is referred to as the 'respiratory burst' (RB) and generates the production of unstable and toxic reactive oxygen intermediates (ROD. These oxidizing species (O2, H 2 0 2, OH-; •O2H) released within the phagolysosome or in extracellular areas, contribute to the microbicidal and tumoricidal activity of the phagocytic cells (Cheson et al., 1976; Hamilton et al., 1987; Neveu, 1986; Walker et al., 1981). Several methods have been used to evaluate the formation of reactive oxygen intermediates and the release of enzymes responsible for these reactions (for review see Adams et al., 1981). The RB of activated phagocytes is related to the emission of light, a phenomenon called chemiluminescence (CL) (Cheson et al., 1976; Walker et al., 1981). In the CL assay, the production of light by activated macrophages can be amplified by the addition of luminol to the cells. This compound interacts with the oxidizing species to produce oaminophthalate and a large amount of light at a peak wavelenght of 425 nm; this light is measured in a luminometer. Recently, it has been shown (Green et al., 1991) that the activated macrophages use a novel biochemical pathway to kill and eliminate several pathogens. This pathway is dependent on Larginine, which is converted to L-citrulline and nitric oxide (NO) by an inductive enzyme, nitric oxide synthase. The reactive intermediate (NO) is an intermediate product with a short life span. It is unstable and reacts readily with oxygen and water to yield more stable products; nitrite (NO 2) and nitrate (NO3). The formation of NO£ and NO 3 is inhibited by a structural analogue of L-arginine, NG-monomethyl-L-arginine (L-NMMA). Nathan and Hibbs (1991) have demonstrated that NO is a cytotoxic effector molecule in the tumoricidal activity of activated macrophages.

Nitrite determination provides a measure of the formation of NO by activated macrophages. Nitrite accumulation in culture supernatants can be measured colorimetrically by the Griess reagent (Ding et al., 1988). The methods described here have used the MALU macrophage cell line for PMA- or zymosan-induced luminol dependent CL and nitrite determination.

2.

Materials

2.1. Equipments and reagents 2.1.1. Equipment CO e incubator Heraeus (Beyneix, Marseilles, France) - Luminometer LKB-Wallac 1251 (LKB Instruments, Orsay, France) -Polystyrene cuvette Clinicon (Boehringer Mannheim, Meylan, France) - Spectrophotometer (Perkin Elmer, 55E, OSI, Paris, France) - Tissue culture flasks: 75 cm / and 25 cm 2 Falcon (Labotecnia, Marseilles, France) -24-well multititer plates: Nunc (Poly-Labo, Strasbourg, France) 2.1.2. Reagents 5-amino-2,3-dihydro-l,4-phthalazinedione (Luminol) (Sigma France, Saint-Quentin, France) Bactopeptone Difco (OSI, Paris, France) Bovine serum albumin (Sigma) - Complete Freund's adjuvant (Difco) - Dimethyl sulfoxide (Prolabo, Paris, France) - F o e t a l calf serum (Boehringer Mannheim, France and Eurobio, Les Ulis, France) Fluid thioglycollate medium (Diagnostic Pasteur, Marnes-la-Coquette, France) -L-glutamine (BioM6rieux, Marcy l'Etoile, France) - Lipopolysaccharide B, E. coli 0111:B4 (LPS) (Difco) Lyophilized guinea pig complement (Diagnostic Pasteur) Hanks' balanced salt solution (HBSS) without phenol red (ICN Flow, Orsay, France)

F. Boudard et al. /Journal of lmmunological Methods 174 (1994) 259-268

Mayer buffer (Diagnostic Pasteur) - N-(1-naphthyl)ethylenediamine, dihydrochloride (Sigma) Penicillin-streptomycin (BioM6rieux) Phorbol 12-myristate 13-acetate (Sigma) - Phosphate balanced solution without Ca 2+ and Mg 2+ (ICN Flow) - Phosphoric acide (Prolabo) - NC-monomethyl-L-arginin e (NMMA) (Sigma) - RPMI 1640 medium with 2.2 g / l N a H C O 3 (Boehringer Mannheim) Sodium nitrite (Sigma) Sulphanilamide (Sigma) - Z y m o s a n (Serva, Le Perray en Yvelines, France) -

-

-

-

2.2. Preparation of culture medium and saline solutions 2.2.1. MALU cells culture medium RPMI 1640 medium containing 2.2 g/1 N a H C O 3, 105 U/1 penicillin, 50 m g / l streptomycin, 5 × 10 -5 M 2-mercaptoethanol, 2 mM glutamine and 10% heat inactivated fetal calf serum; this medium is referred to as complete RPMI 1640 medium.

261

For the test, an aliquot was diluted to 100 n g / m l (final concentration) in HBS solution.

2.3.3. Opsonisation of zymosan 100 mg zymosan were dissolved in 10 ml of sterile distilled water, boiled in a water bath for 30 mn and washed twice with 10 ml of sterile distilled water. This operation was repeated once and after the last washing, the zymosan was resuspended in 10 ml of guinea pig complement diluted in Mayer buffer (2 ml buffer for each lyophilized complement bottle). This solution was incubated at 37°C for 30 min, shaken and washed three times with HBS solution. After the last wash, the opsonised zymosan was diluted in 10 ml of HBS solution and stored in 0.5 ml aliquots at -20°C. For the test, zymosan was used at 1 m g / m l (final concentration). 2.3.4. Thioglycollate broth 29.5 g of thioglycollate powder were dissolved in 1 liter of distilled water, then boiled and sterilized by autoclaving. 2.3.5. Bactopeptone broth 1 g of bactopeptone powder was dissolved in 10 ml isotonic NaCI solution, then boiled and sterilized by autoclaving.

2.2.2. HBS and PBS solutions HBS and PBS solution containing 105 U/1 penicillin and 50 m g / l streptomycin.

2. 4. Preparation of reagents for nitrite determination

2.3. Preparation of reagents for CL determination

2.4.1. Solution A Naphthylethylenediamine was dissolved in distilled water at 1 m g / m l and stored at 4°C.

2.3.1. Luminol A stock solution (10 -3 M) was prepared in the dark as follows: 18 mg of luminol were dissolved in 100 ml sterile isotonic NaC1 solution; then, 1 g of BSA was added and the solution was filtered through 0.22 /zm Millipore filter. The luminol was stored in 2 ml aliquots at - 2 0 ° C in the dark. For the test, luminol was diluted at 10 - 4 M (final concentration) in HBS solution.

2.4.2. Solution B Sulphanilamide was dissolved in 5% phosphoric acid diluted in distilled water to 10 m g / m l and stored at 4°C. These two solutions were stable for six months at 4°C.

3.

2.3.2. PMA A stock solution of 1 m g / m l was prepared in dimethylsulfoxide and stored at - 2 0 ° C in 20 /~1 aliquots. P M A is a potent tumor promoter and must be handled with gloves, mask and glasses.

M e t h o d s

3.1. Cells used 3.1.1. MAL U cell line cultures The M A L U cell line was a gift from Dr. Y. Lombard a n d P. Poindron (D6partement d'Im-

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F. Boudard et al. /Journal of lmmunological Methods 174 (1994) 259-268

munologie et d'Immunopharmacologie, Universit6 Louis Pasteur, U E R Pharmacie, Strasbourg, France). Cells were seeded in complete RPMI 1640 medium (v/v) from a 48 h culture supernatant in 75 cm 2 plastic flasks (Falcon) and incubated at 37°C, in a humidified 5% CO 2, 95% air atmosphere. 3 - 4 days later, macrophages began to multiply on a confluent monolayer of 'mesothelial cells'. The culture supernatant was replaced (30 ml/flask) every 48-72 h with complete RPMI 1640 medium. Under these conditions, the coculture was maintained for 1 month. M A L U ceils could be seeded in 25 cm z plastic flasks (Falcon) in 10 ml of complete RPMI 1640 medium under the same conditions as above. From culture flasks, successive subcultures could be infinitely maintained in 25 or 75 cm 2 flasks with complete RPMI 1640 medium. The number of cells which were recovered from a 48 h culture supernatant was 4 - 8 × 105 cells per ml with a 95% viability.

the cells were resuspended in HBS solution and counted. In resident peritoneal cells, 35% of resident macrophages may be found (Abe et al., 1979). For elicited peritoneal cells, mice were injected 4 days earlier with 0.5 ml of Freund's complete adjuvant or 1 ml of thioglycollate broth or 1 ml of bactopeptone broth. Cells were harvested as above.

3.2. Chemiluminescence determination The culture supernatant from 75 or 25 cm 2 plastic flasks was centrifugated at 800 x g for 5 min and resuspended in 1 ml HBS solution. Cell viability and cell numbers were determined by trypan blue dye exclusion. Cells were adjusted in 0.8 ml HBS solution at the cell number required; then, 100 /zl of luminol (10 -4 M) and 100 tzl of activating agent were added to each tube. The e L , expressed in millivolts (mV), was determined under constant agitation at 37°C in the luminometer. The constant agitation of the vial and temperature of 37°C are two important factors in CL determination (Halstensen et al., 1986). The presence of divalent cations (Ca 2+ and Mg 2÷) in HBS solution was important for a maximal response and the presence of E D T A in solutions is known to inhibit e L .

3.1.2. Resident and elicited peritoneal cells Resident peritoneal cells were obtained by peritoneal washing of B A L B / c mice (female mice, 10-12 weeks olds, Charles River, France) with 5 ml of cold PBS. The cell suspension was centrifugated for 5 min at 800 × g and washed twice with cold PBS solution. After the last wash,

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Fig. 1. PMA-induced chemiluminescence of MALU cells (48 h culture supernatant) compared with resident-, thioglycollate- and peptone-elicited peritoneal cells. The results shown are representative of two independent experiments.

F. Boudard et aL /Journal of Immunological Methods 174 (1994) 259-268 3.3. Nitrite determination

originally plated. Each test was performed in triplicate.

M A L U cells in the supernatant of a 3-day culture were collected and the cells were centrifuged at 800 x g for 5 rain. Macrophages were then seeded into the wells of 24-well multititer plates at a density of 1 x 106 cells per ml of cultured m e d i u m per well. The plates were incubated for 2 h at 37°C in 5% C O : to permit the adherence of the cells. The medium was then removed and the cell layer was washed with a 37°C heated HBS solution. Then, fresh complete R P M I 1640 medium with or without stimulating agents was added. After an appropriate incubation period, the supernatants were collected for nitrite measurement. Immediately before the assay, equal volumes of solution A and B were combined to yield the Griess reagent. For each assay, equal volumes of Griess reagent and supernatant were mixed. This mixture was incubated at room t e m p e r a t u r e for 10 min. N O 2 concentration was determined by measuring the absorbance at 550 nm in a spectrophotometer compared to nitrite standards (1-100 /zM N a N O 2 dissolved in distilled water, using distilled water as a blank). The results were expressed as the concentration of nitrite in nmol per 106 cells

3500

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4. Results and discussion 4.1. Chemiluminescence determination 4.1.1. Comparison o f PMA-induced CL o f M A L U cells and resident or elicited peritoneal cells Fig. 1 shows the PMA-induced CL of M A L U cells, resident and elicited peritoneal cells. M A L U cells secreted a large amount of R O I when comp a r e d with resident or elicited peritoneal cells; in this example, the pic values were: 10.05 x 105 m V for M A L U cells, 1.40 x 105 m V for resident cells, 1.34 X 105 m V for peptone-elicited cells and 3.28 x 105 m V for thioglycollate-elicited cells. It should be noted that peptone-elicited cells showed a similar PMA-induced CL to resident cells even though thioglycollate-elicited cells have a more significant level of CL. When peritoneal cells were elicited by complete Freund's adjuvant, the PMA-induced CL was strongly increased when c o m p a r e d with M A L U cells (Fig. 2; pic values were: 28.55 x 105 m V for elicited cells and 10.55 x 105 m V for M A L U cells).

Freund elicited cells MALUcells

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2

4

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8

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12

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Time (mn)

Fig. 2. PMA-induced chemiluminescence of MALU cells (48 h culture supernatant) and Freund's complete adjuvant elicited-peritoneal cells. The results shown are representative of two independent experiments.

F. Boudard et aL /Journal of Immunological Methods 174 (1994) 259-268

264

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Fig. 3. Influence of cell concentration on the PMA-induced chemiluminescence of MALU cells (48 h culture supernatant). The results shown are representative of two independent experiments.

Taken together, these results show that M A L U cells generated large amounts of ROI which seems to correspond to a 'preactivated' state of the cells.

of cells ranging from 0.125 to 2 × 106 cells per ml: a significant CL response was related to cell numbers when these ranged from 0.5 to 2 × 106 cells per ml. Without any activating agent, M A L U cells did not generate CL. In PMA-induced CL (Fig. 3), the maximal CL intensity was obtained between 2 - 4 min and decreased rapidly. In zymosan-induced CL (Fig. 4), the maximal CL intensity occurred later, up to 40 min, and decreased slowly after a plateau. It should be noted that the pic value obtained with zymosan was lower than with PMA.

4.1.2. Influence of cell number on PMA- and zymosan-induced CL Since cell numbers may be a limiting factor, we evaluated the smallest cell number permitting a significant signal in the CL response to P M A and zymosan. As shown in Figs. 3 and 4, PMA- and zymosan-induced CL depending on the number

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Number of cellslml (x 10 6)

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Fig. 4. Influence of the cell concentration on the zyrnosan opsonised-induced chemiluminescence of MALU cells (48 h culture supernatant). The results shown are representative of two independent experiments.

F. Boudard et aL /Journal of Immunological Methods 174 (1994) 259-268

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Fig. 5. Influence of the incubation time at 37°C on the PMA-induced chemiluminescence of MALU cells (48 h culture supernatant; 7 × 105 cells/ml). The results shown are representative of two independent experiments.

4.1.3. Influence of incubation times on PMA-induced CL of M A L U cells treated with drugs The effect of drugs on M A L U cells could be studied by CL directly or after a pretreatment for different incubation times. When drugs were added simultaneously with P M A at 37°C, CL was measured immediatly and compared to the controls. When the drugs were tested using a pretreatment at 37°C, different times of incubation were chosen depending on the drug tested. To evaluate the influence of the incubation time on PMA-induced CL of M A L U cells, we tested different incubation times on the CL response. These results are shown in Fig. 5: the intensity of CL decreased quickly in proportion to the incubation time. Consequently, when the in~,

cubation time was greater than 30 min, it was necessary to pretreat in the culture flask before performing the CL test. When the drugs were tested using a pretreatment of 1 h at 4°C, the pic value of PMA-induced CL was not significantly modified but appeared later (Fig. 6). A control showing the effect of the drug on the cells without any activating agent was also required.

4.1.4. Effect of LPS pretreatment on the CL of M A L U cells The M A L U cells were seeded in a 25 cm 2 culture flask; on days 3 and 5, the supernatants were replaced with complete RPMI 1640 medium.

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Fig. 6. Influence of the incubation time at 4°C on the PMA-induced chemiluminescence of MALU cells (48 h culture supernatant; 7 × 105 ceils/ml). The results shown are representative of two independent experiments.

F. Boudard et al. /Journal of Immunological Methods 174 (1994) 259-268

266

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Fig. 7. Effect of a 24 h pretreatment with LPS on the PMA-induced chemiluminescence of MALU cells (5X 105 cells/ml). The results are derived from the CL response over 546 s (mean+SD, n = 7-14). *P < 0.05 by Student's t test. The LPS (1 n g / m l and 100 n g / m l ; final concentrations) was added to the culture flasks at day 5 and the CL was determined 24 h later. Fig. 7 shows the effect of LPS p r e t r e a t m e n t on the PMA-induced CL of M A L U cells; the endotoxin significantly enhanced the CL response compared to the control cells. The fact that M A L U cells respond to a low concentration of LPS (1 n g / m l ) confirmed that this cell line was in a 'preactivated state'; generally, the concentrations of LPS used to activate macrophages, were in a range of 0.1 / ~ g / m l to 1 0 / ~ g / m l .

4.1.5. Expression of results The signal was measured in mV. W h e n the cells were stimulated with a potent activator (PMA), the pic value was sufficient to express the results. W h e n the cells were stimulated with zymosan or when the intensity of the signal was weak, results were expressed as the integration of the signal under the curve. The percent change in the CL response is defined as follows: % = ( ( p e a k value (or integrate) of treated cells) / ( p e a k value (or integrate) of untreated cells) - 1) x 100

4.1.6. Advantages and disadvantages of CL determinations using M A L U cells CL was found to be a rapid and easy method which measured the generation of R O I from

phagocytic cells after appropriate activation; R O I have been found in different phagocytic cells in man and other mammalian species. When cells are not in an 'activated state', as resident macrophages, these cells secrete low amounts of R O I and the CL assay requires a large n u m b e r of cells (106-107 per ml) to obtain a significant response. In the case of M A L U cells, the cell numbers which are required for each determination are lower ( < 106) because these cells are in a 'preactivated state'. This preactivated state can be explained, in part by the presence of IFN-7 in culture supernatants. This factor is known to induce the priming of macrophages. However, the IFN-7 producing cells in this coculture are not identified (Hamilton et al., 1987; Lombard et al., 1988). Moreover, under appropriate culture conditions (supernatants of 48-72 h from a 75 cm 2 culture flask), a large number of cells can be obtained for CL assay. We have observed some growth and cell number variation when the supernatants were recovered from cultures using different lots of fetal calf serum; it is also important to verify the efficiency of the serum employed. During the culture and infinite subcultures of M A L U cells, it is important to confirm the absence of LPS and mycoplasma contamination both of which are able to activate M A L U cells. It is also important to bear in mind that during the culture of M A L U cells, unknown growth factors or activating substances could be released into the supernatant, thereby modifying the response of the cells.

4.2. Nitrite determination 4.2.1. Effect of LPS and of a (1 ~ 3)-fl-o-glucan on nitrite determination of M A L U cells Fig. 8 shows the LPS-induced nitrite production of M A L U cells. The LPS resulted in a statistically significant NO~- production after a 48 h incubation period. After this time, the N O ~ liberation increased and reached a plateau between 120 and 144 h. For example, Fig. 9 shows the kinetics of N O 2 synthesis in response to a (1 ~ 3)-/3-o-glucan for M A L U cells. A 48 h phase was followed by a

F. Boudard et al. /Journal of Immunological Methods 174 (1994) 259-268 35

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linear increase in NO~- for 144 h and NO 2 production was statistically significant after 72 h. Under our experimental conditions, M A L U cells were able to produce large amounts of RNI in response to different stimuli.

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4.2.2. Specificity of nitrite production by MALU

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cells 48 h

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96 h Incubation

120 h

144 h

time

Fig. 8. Effect of LPS on nitrite production by M A L U cells during different incubation periods (mean + SD, n = 2-4). * P < 0.05 by Student's t test.

35

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267

In order to verify the specificity of nitrite production by M A L U cells, we examined the effect of L-NMMA, a selective inhibitor of the Larginine-dependent pathway. As shown in Fig. 10, L-NMMA significantly inhibited the NO 2 release by LPS-stimulated M A L U cells; it should be noted that L-NMMA inhibited the basal NO 2 secretion of M A L U cells.

4.2.3. Expression of results

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Each test was carried out in triplicate; the results are expressed as the mean of the absorbance + standard deviation (SD). The nitrite concentration was calculated by reference to a standard curve (NaNO2) and expressed in terms of nmol NO~- per 10 6 cells.

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Fig. 9. Effect of (1 ~ 3)-/3-D-glucan on nitrite production by M A L U cells over different incubation times (mean + SD, n = 3-11). * P < 0.05 by Student's t test.

4.2.4 Advantages and disavantages of N O r determination using MALU cells Nitrite determination by the Griess reaction is a simple and rapid. It is also flexible and may be adapted easily in the laboratory. For example; after an appropriate incubation period, the cup

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Fig. 10. Effect of N M M A on nitrite production by M A L U cells either used alone or stimulated with LPS over different incubation periods. The results shown are representative of three i n d e p e n d e n t experiments (mean + SD, n = 3).

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F. Boudard et al. /Journal of Immunological Methods 174 (1994) 259-268

ture supernatants can be stored at -80°C for subsequent testing. Likewise, this assay has been adapted to microtiter plates and absorbance measured at 550 nm in a microELISA reader. The measurement is then significantly faster. This method gives reproducible and significant results but it is important to note that variability in nitrite production can be observed depending on the fetal calf serum used. 5. Conclusion

The chemiluminescence and nitrite determinations are rapid, easy and inexpensive methods which measure indirectly the tumoricidal and microbicidal activities of activated macrophages. The induction of ROI and RNI appear to be independent mechanisms although the biochimical pathways of these metabolic reactions are not completely known (Ding et al., 1988; Mau~l et al. 1991). Indeed, the release of ROI (CL determination) by macrophages is rapid after appropriate activation (< 1 h) whereas the release of RNI (nitrite determination) requires a longer incubation period to detect a significant NO 2 amount in the culture medium (> 24 h). The culture conditions of MALU cells provide large numbers of ceils in a 'preactivated' state. These cells can then be used to test the effects of immunotherapeutic drugs on the ROI and RNI production and also investigated the biochemical pathways involved in the activation of macrophages under appropriate stimulation. References Abe, K., Honma, S. and Ito, T. (1979) Peritoneal cells in mice: quantitative and qualitative cell morphology. Am. J. Anat., 156, 37.

Adams, D.O. et al. (Eds.) (1981) Methods for Studying Mononuclear Phagocytes. Academic Press, New York, pp. 477-510. Cheson, B.D., Christensen, R.L., Sperling, R., Kohler, B.E. and Babior, B.M. (1976) The origin of chemiluminescence of phagocytosing granulocytes. J. Clin. Invest. 58, 789. Ding, A.H., Nathan, C.F. and Stuehr, D.J. (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal maerophages. Comparison of activating cytokines and evidence for independent production. J. Inununol. 141, 2407. Falkenberg, U., Lombard, Y., Giaimis, J., Poindron, P. and Falkenberg, F.W. (1992) Phenotypic characterization of three long-term cultured murine resident macrophages lines. Res. Immunol. 143, 25. Green, S.J., Nacy, C.A. and Meltzer, M.S. (1991) Cytokine-induced synthesis of nitrogen oxides in macrophages: a protective host response to Leishmania and other intracellular pathogens. J. Leukoc. Biol. 50, 93. Halstensen, A., Haneberg, B., Glette, J., Sandberg, S. and Solberg, C.O. (1986) Factors important for the measurement of chemiluminescence production by polymorphonuclear ieukocytes. J. Immunol. Methods 88, 121. Hamilton, T.A. and Adams, D.O. (1987) Molecular mechanisms in signal transduction in macrophages. Immunol. Today 8, 151. Lombard, Y., Bartholeyns, J., Chokri, M., Illinger, D., Hartmann, D., Dumont, S., Kaufmann, S.K.E., Landmann, R., Loor, F. and Poindron, P. (1988) Establishment and characterization of long-term cultured cell lines of murine resident macrophages. J. Leukocyte Biol. 44, 391. Mau~l, J., Betz Corradin, S. and Buchmuller Rouiller, Y. (1991) Nitrogen and oxygen metabolites and the killing of Leishmania by activated murine macrophages. Res. Immunol. 142, 577. Nathan, C.F. and Hibbs, J.B. (1991) Role in nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3, 65. Neveu, P.J. (1986) The mononuclear phagocyte system. Bull. Inst. Pasteur 84, 23. Walker, E.B., Van Epps, D.E. and Warner, N.L. (1981) Macrophage chemiluminescence. In: H.B. Herscowitz, H.T. Holden, J.A. Bellanti and A. Ghaffar (Eds.), Manual of Macrophage Methodology. Collection Characterization and Function. Marcel Dekker, New York, p. 389.