Nitric oxide synthase activity and mRNA expression in chicken macrophages

Nitric Oxide Synthase Activity and mRNA Expression in Chicken Macrophages1 I. HUSSAIN and M. A. QURESHI2 Department of Poultry Science and Graduate Program of Immunology, North Carolina State University, Raleigh, North Carolina 27695-7608 completely abolished nitrite production. The addition of 10% vol/vol lymphokines exhibited an additive effect on nitrite production in conjunction with LPS. The increased nitrite production by the K-strain and MQNCSU macrophages corresponded to an increased expression of iNOS mRNA as compared to the mRNA produced by GB1 and GB2 macrophages. The iNOS mRNA kinetics study revealed that mRNA levels peaked between 6 to 12 h. The cells from avian lymphoid lineage failed to produce any detectable iNOS activity. These studies showed that macrophages from varying sources differ in NOS activity and implied that genetic background may dictate the extent of argininemediated contribution in various biological and immunological functions.

(Key words: nitric oxide synthase, macrophages, L-arginine, chicken) 1997 Poultry Science 76:1524–1530

INTRODUCTION Macrophages play a key role in innate and adaptive immunity. Being phagocytic in nature, these cells initiate an immune response by antigen processing and presentation to T- and B-lymphocytes. Furthermore, macrophages secrete an array of cytokines, such as interleukin1 (IL-1) and IL-6, and other immunologically important effector biomolecules, such as reactive oxygen intermediates (Unanue and Allen, 1987; Golemboski et al., 1990; Sung et al., 1991; Qureshi et al., 1994). Recently, a newly discovered cytotoxic entity, nitric oxide (NO), has been shown to play a pivotal role in the innate immune functions of macrophages (Schmidt and Walter, 1994). Macrophages produce NO by oxidizing the guanidino nitrogen of L-arginine by an enzyme, nitric oxide

Received for publication February 13, 1997. Accepted for publication June 9, 1997. 1Salaries and research support provided by state and federal funds appropriated to the North Carolina Agricultural Research Service, North Carolina State University. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, nor criticism of similar products not mentioned. 2To whom correspondence should be addressed.

synthase (NOS) (Marletta, 1994). This enzyme is inducible in macrophages by bacterial endotoxins such as lipopolysaccharide (LPS), or by lymphokines (Nathan, 1992; Hauschildt et al., 1990) and is termed as inducible NOS (iNOS). In contrast, the constitutively expressed isoform, cNOS, is expressed in cells such as endothelial cells and neurons (Nathan, 1992). In the murine system, both the cNOS and iNOS isoforms have been cloned and their activities examined extensively (Nathan, 1992). Being a very short-lived radical, NO is converted into more stable products such as nitrite (NO2) and nitrate (NO3) (Stuehr and Marletta, 1985, 1987). Evidence from several fields of research has suggested the involvement of this pathway in the macrophage-tumoristatic and microbiostatic effector mechanisms (Iyengar et al., 1987; Stuehr and Nathan, 1989; Adams et al., 1990; Suk et al., 1993). In contrast to mammals, chickens can not synthesize L-arginine (Riley et al., 1986), and hence fully depend on the exogenous source of L-arginine for metabolic pathways requiring L-arginine. Taylor et al. (1992) have shown that dietary L-arginine influences the outcome of Rous sarcoma-induced tumors in chickens. This requirement suggests that iNOS activity in terms of L-argininemediated metabolites may be important immunologically in chickens. The objectives of this study were to

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ABSTRACT The activity of inducible nitric oxide synthase (iNOS) enzyme was quantified in chicken macrophages. Macrophages from Cornell K-strain (B15B15), GB1 (B13B13), and GB2 (B6B6) chickens and a transformed cell line (MQ-NCSU) were incubated with or without varying concentrations of bacterial lipopolysaccharide (LPS). The culture supernatants were tested for the presence of nitrite. Macrophages from either source produced minimal nitrite (< 4.4 mM/1 × 106 cells) levels without LPS stimulation. However, nitrite levels produced by K-strain (42 mM) and MQNCSU (41 mM) macrophages were higher (P < 0.05) than those produced by the GB1 (14 mM) and GB2 (14 mM) per 1 × 106 macrophages with optimum LPS concentration range of 50 ng to 1 mg/mL. The addition of an Larginine analog, NGMMLA, at a concentration of 200 mM

NITRIC OXIDE SYNTHASE ACTIVITY

examine iNOS activity as measured by nitrite production and mRNA expression in chicken macrophages with different genetic backgrounds.

MATERIALS AND METHODS

Cell Source

Abdominal Exudate Cell Collection Abdominal exudate cells (AEC) were collected as previously described (Qureshi et al., 1986). Briefly, a 3% (wt/vol) Sephadex suspension was prepared by preswelling Sephadex G-503 superfine powder in autoclaved, distilled water overnight. After two washes with distilled water, Sephadex was resuspended in sterile 0.85% NaCl solution, and chickens were injected intra-abdominally with the Sephadex suspension at a dose of 1 mL/100 g body weight. The abdominal exudate from each chicken was collected 42 h post-Sephadex injection by abdominal lavage with 30 mL per bird of cold 0.85% NaCl containing 0.5 U/mL of heparin. The AEC suspension was kept on ice in siliconized tubes for about 15 min to allow any residual Sephadex beads to settle. Supernatants containing cells were then decanted to new siliconized tubes. Cells were pelleted by centrifugation (350 × g for 10 min) and washed once with low endotoxin (< 2 pg/mL) RPMI-16404 (without phenol red) supplemented with 5% fetal calf serum (FCS) and 100 mg/mL of streptomycin and 2 mg/ mL of fungizone (complete medium) before further treatment. This Sephadex-elicitation protocol yields greater than 95% adherent macrophages in AEC (Qureshi et al., 1986).

Cell Lines All cell lines (MQ-NCSU, RP9, and CU14) were maintained in LM-Hahn’s growth medium (Qureshi et al.,

3Sigma Chemical Co., St. 4Lineberger Tissue Culture

1990) at 41C in a 5% CO2 humidified incubator. For experimental endpoints, cells were collected in their log phase of growth, washed with RPMI (without phenol red) complete medium, and then resuspended at a final concentration of 1 × 106 viable cells per milliliter. The viability of cells was determined by the trypan blue exclusion method (Phillips, 1973).

Culture Conditions for Nitrite Assay Pooled AEC from four to five chickens per strain and MQ-NCSU cells were seeded in a 24-well culture plate5 at a cell concentration of 1 × 106 per milliliter of complete medium per well. The cell viability was determined for each sample prior to setting up experimental cultures and was found to be greater than 98% at the time of seeding. Culture plates containing AEC were incubated for 2 h at 41 C, 5% CO2 to allow macrophage adherence. After incubation, the nonadherent cells were removed by gentle washing with the complete medium. Culture wells containing adherent AEC or MQ-NCSU cells were incubated in 1 mL of the complete medium containing different concentrations of lipopolysaccharide (LPS, Escherichia coli O.55.B5)6 and, if present, 10% (vol/vol) of lymphokine (LK) provided by H. S. Lillehoj, USDA, Beltsville, MD 20705. The cells were further incubated for a period of 24 h, at which point the culture supernatants were collected for nitrite analysis. A guanidino methylated derivative of L-arginine, NG monomethyl-Larginine (NGMMLA), a competitive inhibitor of the Larginine-dependent effector pathway (Stuehr and Marletta, 1987; Adams et al., 1990) was also employed in some experiments to study its effect on nitrite synthesis by chicken macrophages.

Nitrite Assay Nitrite levels were measured in 96-well microtiter plates by mixing 100 mL of the macrophage culture supernatant with an equal volume of Griess reagent (one part 0.1% naphthylethylenediamine dihydrochloride to one part 1% sulfanilamide in 5% phosphoric acid) as described by Green et al. (1982). After 10 min incubation at room temperature, the change in color indicative of nitrite presence was quantified by reading the plates at A540 on an ELISA plate reader.7 An average of three measurements per sample was used in final analysis. A standard curve of optical densities (OD) at A540 was generated using various concentrations of sodium nitrite dissolved in RPMI-1640 growth medium (without phenol red). Nitrite levels of the culture supernatants were determined by comparing their OD values with that of the standard.

RNA Isolation and Northern Blot Analysis for iNOS Louis, MO 63178-9916. Facility, University of North Carolina-

Chapel Hill, NC 27599. 5Corning, NY 14831. 6Difco Laboratories, Inc., Detroit, MI 48232. 7Bio-Rad Laboratories, Richmond, CA 94804.

The cells from different sources were added in 25 cm2 culture flasks at a concentration of 1 × 106 viable cells per milliliter in 15 mL of complete medium. After a 2-h incubation period, the medium was changed to

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Abdominal exudate cells were collected from 3- to 5-wk-old Cornell K-strain (B15B15), GB1 (B13B13), and GB2 (B6B6) chickens maintained at the Department of Poultry Science, North Carolina State University. The chickens were fed a corn-soybean diet consisting of 17% crude protein. Feed and water were available for ad libitum access. The MQ-NCSU, a transformed chicken macrophage cell line (Qureshi et al., 1990) comprised another myeloid lineage cell source. For comparison, cells of the lymphoid lineage, namely LSCC-RP9 (B15B2), a chicken B cell line (Okazaki et al., 1980), and MDCC-CU14 (B19C2), a chicken T cell line (Calnek et al., 1981), were also examined for nitrite production and iNOS mRNA expression.

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without LPS stimulation. However, when cultured with increasing concentrations of LPS, a corresponding increase in nitrite production was observed. Macrophages from K-strain chickens exhibited an overall heightened nitrite synthesis as compared to the macrophages from GB1 and GB2 chickens at every LPS concentration from 1 ng to 3 mg/mL (P < 0.05). At LPS concentrations of 50 ng/ mL and beyond, K-strain macrophages produced a peak level of nitrite ranging from 40 to 42 mM/106 cells. This peak level of nitrite at the same 50 ng/mL LPS concentration was, however, significantly lower in GB1 and GB2 macrophages (13 to 15 mM) (P < 0.05). At any given LPS concentration, GB1 and GB2 macrophages did not differ from each other in nitrite synthesis (P < 0.05). The MQ-NCSU cell line macrophages produced higher nitrite levels (P < 0.05) at 10 ng/mL of LPS and beyond than the GB1 and GB2 macrophages. Although K-strain macrophages produced higher nitrite levels at 1 and 10 ng/mL of LPS, at LPS concentrations of 50 ng and beyond, MQ-NCSU macrophages produced nitrite comparable to the K-strain chicken macrophages. In order to show the specificity of L-arginine-based nitrite production, an L-arginine analog, NGMMLA, was added to macrophages cultures in varying concentrations in the presence of 1 mg/mL of LPS. The amounts of nitrite produced by K-strain and MQ-NCSU macrophages declined with the increasing concentration of NGMMLA in culture (data not shown). At 200 mM, NGMMLA completely blocked nitrite production down to the unstimulated macrophage levels.

Statistical Analysis Nitrite data from LPS and LPS plus LK-stimulated macrophages were analyzed using one- and two-way analysis of variance, respectively (SAS Institute, 1989). Individual effects of LPS or LK were compared using Duncan’s multiple range test at an a level equal to 0.05.

RESULTS

Nitrite Production: Macrophages vs LPS Figure 1 shows the nitrite production by macrophages from different sources when stimulated with various LPS concentrations. Macrophages from either source produced minimal levels of nitrite (< 4.4 mM/1 × 106 cells)

8Life Technologies, Grand Island, NY 14072-0068. 9Schleicher and Schuell, Keene, NH 03431. 10Boehringer Mannheim, Indianapolis, IN 46250-0414. 11Tropix, Bedford, MA 01730. 12Oncor, Gaithersburg, MD 20877.

FIGURE 1. Nitrite production by chicken macrophages. Sephadexelicited abdominal exudate cells from Cornell K-strain, GB1, GB2, and a transformed macrophage chicken cell line, MQ-NCSU, were stimulated with the indicated concentrations of lipopolysaccharide (LPS) for 24 h and the supernatants were analyzed for nitrite levels. Each data point represents a pooled mean nitrite concentration (± SD) from three separate experiments. Each experiment consisted of two to three replicatecultures per LPS concentrations.

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medium with or without LPS (1 mg/mL) and cells were reincubated up to 24 h. After collecting supernatant (for nitrite analysis), total RNA was isolated by lysing the cells in Trizol Reagent8 following the manufacturer’s protocol. For Northern blot analysis, samples (20 mg of total RNA per lane) were separated through a 1.2% agaroseformaldehyde gel by electrophoresis and transferred to Nytran Plus membrane9 using 20 × SSC (175.3 g sodium chloride, 88.2 g sodium citrate/L; pH 7.0) as described by Sambrook et al. (1989). The RNA was cross-linked to the membrane by ultraviolet (UV) irradiation at 302 nm for 3 min with a UV transilluminator. The blots were prehybridized for 4 h at 45 C in SDS buffer {50% formamide, 7% SDS, 5 × SSC, 2% blocking reagent,10 50 mM sodium phosphate (pH 7.0), 0.1% N-lauroylsarcosine}. The chicken iNOS mRNA was detected using a 4.5-kb chicken iNOS cDNA probe (Genbank Accession Number U 46504) provided by C. C. McCormick (Cornell University). The iNOS cDNA was labeled with digoxigenin following the vendor’s protocol.11 The hybridization was carried out with 400 ng of the labeled iNOS cDNA probe at 45 C overnight in 10 mL of prehybridization solution. After hybridization, the blots were washed twice (20 min each) with 2 × SSC, 0.1% SDS at 45 C and then twice with 0.5 × SSC, 0.1% SDS at 68 C for 20 min each. The hybridized probe was immunodetected with anti-digoxigenin-AP Fab fragments 11 using disodium 2-chloro-5-(4methoxyspiro{1,2-dioxetane-3,2-(5-chloro) tricyclo [3.3.1.13,7] decan}-4-yl)-1-phenyl phosphate (CDP Star)11 as a chemiluminescent substrate following manufacturer’s instructions. The lumigraphs were developed after a 20-s exposure and were examined visually. Digoxigenin-labeled chicken b-actin cDNA12 was used as a house-keeping gene control.

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NITRIC OXIDE SYNTHASE ACTIVITY TABLE 1. Nitrite production by chicken macrophages after stimulation with various concentrations of lipopolysaccharide (LPS) and 10% (vol/vol) lymphokine (LK) mixture1 Stimuli

Macrophage source

LK

LPS

MQ-NCSU

(%) 10 10 10 10 10 10 . . . 10

(ng/mL) 10 25 300 500 1,000 5,000 1,0003 None

109 112.4 112.9 108.7 114 117.1 41 60.3

Cornell K-strain

GB1

GB2

(mM)2 ± ± ± ± ± ± ± ±

2.3 3.4 3.2 2.9 5.2 4.6 3.7 3.6

110.97 107.08 113.3 111.7 113.7 115 43.1 76.8

± ± ± ± ± ± ± ±

6.3 5.4 4.8 4.4 5.2 6.8 3.2 7.4

73.9 75.6 77.8 77.3 82.3 85.3 14 51.6

± ± ± ± ± ± ± ±

6.7 7.1 3.6 3.2 2.6 3.2 0.6 3.7

78.3 80.7 82.3 84.4 89.5 94.3 14 49.6

± ± ± ± ± ± ± ±

5.1 4.3 3.6 4.7 4.3 3.2 1.7 4.3

Nitrite Production: Macrophages vs LPS and Lymphokines

Northern Blot Analysis of iNOS mRNA

Table 1 shows the nitrite levels when macrophages from various sources were stimulated with various concentrations of LPS in the presence of 10% (vol/vol) lymphokines. The addition of LK in conjunction with every LPS concentration caused an over twofold increase in nitrite levels produced by K-strain and MQ-NCSU macrophages as compared with the LPS-alone (1 mg/mL) treated cultures. However, a greater increase (five- to sixfold) was observed in GB1 and GB2 macrophages in LPS plus LK-treated culture supernatants over 1 mg/mL LPS alone. Interestingly, stimulation with LK alone resulted in higher nitrite levels by every macrophage source than stimulation with LPS alone. Furthermore, similar to the results shown in Figure 1, GB1 and GB2 macrophages were low responders in nitrite production even with LPS plus LK stimulation as compared with the K-strain and MQ-NCSU macrophages.

The first series of experiments was designed to study the kinetics of iNOS mRNA expression after stimulation with LPS (1mg/mL). Total RNA was extracted from MQNCSU cells at different time points (Figure 2) after LPS challenge and 20 mg of total RNA per lane was used for Northern blot analysis. As seen in Figure 2, detectable levels of iNOS mRNA were observed 2 h after LPS stimulation. These levels peaked between 6 to 12 h and by 24 h iNOS mRNA levels had declined. Nitrite levels in the supernatants of these cultures were, however, detectable after 4 h of LPS stimulation and accumulated over time such that maximum nitrite production was observed at 24 h after LPS stimulation (data not shown). Because the variation in nitrite levels described in Figure 1 was dependent upon the source of macrophages, we isolated total RNA from MQ-NCSU, K-strain, GB1, and GB2 macrophages after LPS (1 mg/mL) stimulation. Total RNA was then analyzed to determine the relative

FIGURE 2. Kinetics of inducible nitric oxide synthase (iNOS) mRNA in activated MQ- NCSU cell line. Total RNA from MQ-NCSU cell line was collected at 0, 2, 4, 6, 8, 10, 12, and 24 h (Lanes 0 to 7, respectively) after LPS (1 mg/mL) treatment. Twenty micrograms total RNA per lane was run through 1.2% formaldehyde agarose gel by electrophoresis. After Northern blotting, the membrane was probed with a chicken iNOS specific cDNA. This figure is representative of three separate experiments yielding identical iNOS (4.5 kb) expression kinetics.

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11 × 106MQ-NCSU and Sephadex-elicited abdominal macrophages from three lines of chickens were exposed to various LPS concentrations with 10% (vol/vol) LK for 24 h. 2The data are the mean ± SD of micromoles of nitrite from a pool of two separate experiments. In each experiment, the nitrite levels were determined from triplicate wells at each given concentration. 3No LK mixture was added.

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DISCUSSION Recent reports have described various aspects of macrophage-mediated and L-arginine-dependent production of reactive nitrogen intermediate such as nitric oxide, nitrite, and nitrate by chicken macrophages (Sung et al., 1991; Qureshi et al., 1994). The study reported here further extends chicken NOS biology in terms of its expression and activity. It is clear from the data presented that the Sephadex-elicited chicken abdominal exudate macrophages or transformed chicken

FIGURE 3. Relative abundance of inducible nitric oxide synthase (iNOS) mRNA in macrophages from different sources: Macrophages were stimulated with 1 mg/mL lipopolysaccharide (LPS) for 24 h and total RNA extracted. Northern blot was prepared using 20 mg per lane of total RNA for iNOS (4.5 kb) mRNA analysis from LPS stimulated or unstimulated MQ-NCSU cell line (Lanes 1 to 2), GB1 (Lanes 3 to 4), Kstrain (Lanes 5 to 6), and GB2 (Lanes 7 to 8) macrophages. Chicken bactin cDNA (2 kb) was used as a house-keeping gene control. This figure is a representative of three separate experiments yielding similar results.

macrophage cell line require exogenous stimuli such as LPS or LK to produce detectable levels of nitrite in culture supernatants. Furthermore, chicken macrophages utilize L-arginine for this reaction as the addition of NGMMLA, an arginine analog, inhibited nitrite levels in a dose-dependent manner. Likewise, Sung et al. (1994) have shown that NO-induced mitochondrial injury can be blocked by L-arginine analog. At a given LPS concentration, (e.g., 50 ng/mL) and constant cell number, the data show a clear difference in the ability of macrophages from different genetic sources to produce nitrite (Figure, Table 1). Based on these data, it is reasonable to state that the GB1 (B13B13) and GB2 (B6B6) chickens are hyporesponsive to LPS as compared to the Cornell K-strain (B15B15) chickens and MQ-NCSU (macrophage cell line of broiler origin with unknown MHC haplotype) macrophages. These findings are possible avian examples of a similar phenomenon reported in murines, in which C3H/HeJ mice were shown to be hyporesponsive to LPS (Stuehr and Marletta, 1987). Interestingly, the nitrite levels of macrophages from all genetic sources were comparable when stimulated with LK alone (Table 1). This suggests that the low responsiveness in GB1 and GB2 chickens for iNOS activity is presumably due to a defect in LPS-mediated signal transduction pathway. The addition of LK to LPStreated macrophages reduced the level of LPS unresponsiveness in GB1 and GB2 macrophages (Table 1), thereby suggesting a possible LK-mediated compensatory mechanism in macrophage activation for iNOS synthesis and activity. This report, therefore, provides the first evidence that the genetic background may influence the NOS activity in terms of nitrite production within the avian species. To further confirm the NOS activity-dependent genetic differences in arginine metabolism, expression of iNOS mRNA was examined. These experiments further confirmed that the difference in the nitrite levels of macrophage cultures are indeed due to differential expression of iNOS mRNA. The low nitrite producing macrophages (e.g., GB1 and GB2) had lower iNOS mRNA expression relative to the iNOS produced by macrophages from high responders (i.e., K-strain and MQ-NCSU cell line) at 24 h poststimulation. The iNOS mRNA expression in MQ-NCSU cell line peaked between 8 and 12 h after LPS stimulation, which is comparable to the levels reported by Lin et al. (1996) in HD11 chicken macrophage cell line. The reason for selecting 24 h poststimulation time point for nitrite and mRNA comparisons was based on several studies showing that the accumulation of nitrite in culture follows a noticeable lag period to allow iNOS mRNA to translate into functionally active enzyme (Stuehr and Marletta, 1987; Ding et al., 1988). The bases of such differences in iNOS activity amongst macrophages from various sources are not known. Xie et al. (1992) have reported that the increase in the iNOS mRNA in murine macrophages is transcriptional in nature. It is therefore

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abundance of the iNOS mRNA and to see whether any correlation exists between the observed nitrite levels and the mRNA expression. Figure 3 shows the relative abundance of iNOS mRNA in MQ-NCSU, GB1, K-strain, and GB2 chicken macrophages. As can be seen, a 4.5-kb iNOS band was detectable in all macrophage samples only after LPS stimulation. Macrophages from MQ-NCSU cell line and K-strain chickens exhibited high intensity bands indicative of higher iNOS mRNA synthesis than that of the macrophages from GB1 and GB2 chickens. The b-actin transcript was comparable in all lanes except MQ-NCSU, which always expressed little or no detectable b-actin message (Unpublished data). These differences in iNOS mRNA expression, therefore, followed a similar macrophage-source-dependent nitrite production profile as shown in Figure 1. The last series of experiments was conducted to compare iNOS mRNA expression by cells of myeloid (MQ-NCSU and Cornell K-strain macrophages) vs lymphoid (RP-9, CU-14: lymphoblastoid cell line) lineage. Cells were stimulated with 1 mg/mL LPS and total RNA was extracted and analyzed by Northern blot. The cells of lymphoid lineage did not express iNOS mRNA, whereas cells of the myeloid lineage expressed a 4.5-kb band corresponding to iNOS mRNA (data not shown).

NITRIC OXIDE SYNTHASE ACTIVITY

ACKNOWLEDGMENTS The authors thank S. J. Lamont of Iowa State University for providing GB1 and GB2 strains of chickens. Secretarial assistance by J. Seale and technical assistance by R. Ali is greatly appreciated.

REFERENCES Adams, L. B., J. B. Hibbs, Jr., R. R. Taintor, and J. L. Krahenbuhl, 1990. Microbiostatic effect of murineactivated macrophages for Toxoplasma gondii. J. Immunol. 144:2725–2729. Bacon, L. D., R. L. Witter, L. B. Crittenden, A. M. Fadly, and J. Motta, 1981. B-haplotype influence on Marek’s disease, Rous sarcoma, and lymphoid leukosis virus-induced tumors in chickens. Poultry Sci. 60:1132–1139. Briles, W. E., R. W. Briles, R. E. Taffs, and H. A. Stone, 1983. Resistance to a malignant lymphoma in chickens is mapped to subregion of major histocompatibility (B) complex. Science 219:977–979. Calnek, B. W., W. R. Shek, and K. A. Schat, 1981. Spontaneous and induced herpes virus genome expression in Marek’s disease tumor cell lines. Infect. Immun. 34:483–491. Chartrain, N. A., D. A. Geller, P. P. Koty, N. F. Sitrin, A. K. Nussler, E. P. Hoffman, T. R. Billiar, N. I. Hutchinson, and J. S. Mudgett, 1994. Molecular cloning, structure, and

chromosomal localization of the human inducible nitric oxide synthase gene. J. Biol. Chem. 269:6765–6772. Ding, A. H., C. F. Nathan, and D. J. Stuehr, 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. Immunol. 141:2407–2412. Golemboski, K. A., J. Whelan, S. Shaw, J. E. Kinsella, and R. R. Dietert, 1990. Avian inflammatory macrophage function: Shifts in arachidonic acid metabolism, respiratory burst, and cell surface phenotype during the response to Sephadex. J. Leukocyte Biol. 48:495–501. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum, 1982. Analysis of nitrate, nitrite and [15N] nitrite in biological fluids. Anal. Biochem. 126:131–138. Hauschildt, S., E. Bassenge, W. Bessler, R. Busse, and A. Mulsch, 1990. L-arginine-dependent nitric oxide formation and nitrite release in bone marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide. Immunology 70:332–337. Iyengar, R., D. J. Stuehr, and M. A. Marletta, 1987. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: Precursors and role of the respiratory burst. Proc. Natl. Acad. Sci. USA. 84:6369–6373. Lamont, S. J., C. Bohn, and N. Cheville, 1987. Genetic resistance to fowl cholera is linked to the major histocompatibility complex. Immunogenetics 25:284–289. Lillehoj, H. S., M. D. Ruff, L. D. Bacon, S. J. Lamont, and T. K. Jeffers, 1989. Genetic control of immunity to Eimeria tenella. Interaction of MHC genes and non-MHC linked genes influence levels of disease susceptibility in chickens. Vet. Immunol. Immunopathol. 20:135–148. Lin, A. W., C. C. Chang, and C. C. McCormick, 1996. Molecular cloning and expression of an avian macrophage nitric oxide synthase cDNA and the analysis of the genomic 5′-flanking region. J. Biol. Chem. 271: 11911–11919. Marletta, M. A., 1994. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78:927–930. Nathan, C., 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6:3051–3064. Okazaki, W., R. L. Witter, C. Romero, K. Nazerian, J. M. Sharma, A. Fadly, and D. Ewert, 1980. Induction of lymphoid leukosis transplantable tumors and the establishment of lymphoblastoid cell lines. Avian Pathol. 9: 311–329. Phillips, H. J., 1973. Dye exclusion tests for cell viability. Page 406 in: Culture Methods and Applications. P. F. Kruse, Jr., and M. K. Patterson, Jr., ed. Academic Press, New York, NY. Qureshi M. A., L. Miller, H. S. Lillehoj, and M. D. Ficken, 1990. Establishment and characterization of a chicken mononuclear cell line. Vet. Immunol. Immunopathol. 26: 237–250. Qureshi, M. A., J. A. Marsh, R. R. Dietert, Y.-J. Sung, C. Nicolas-Bolnet, and J. N. Petitte, 1994. Profiles of chicken macrophage effector functions. Poultry Sci. 73:1027–1034. Qureshi, M. A., R. R. Dietert, and L. D. Bacon, 1986. Genetic variation in the recruitment and activation of chicken peritoneal macrophages. Proc. Soc. Exp. Biol. Med. 181: 560–568. Riley, M. M., Jr., E. Esteve-Garcia, and R. E. Austic, 1986. Intestinal absorption of glucose and amino acids in chickens administered monensin. Poultry Sci. 65: 2292–2298.

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possible that the differential expression of iNOS mRNA may be due to 1) differential transcriptional potential (i.e., sensitivity of iNOS regulatory sequences such as NF-kB); 2) iNOS mRNA stability; or 3) differential transcriptional rate. In fact, studies with murine (Xie et al., 1994) and chicken (Lin et al., 1996) macrophages have shown that iNOS expression can be blocked if macrophages are treated with pyrrolidine dithiocarbamate (PTDC), a NF-kB inhibitor. Our studies have further shown that chicken lymphoid lineage cells such as Band T-lymphocytes do not express iNOS, an observation similar to the ones reported in mice (Stuehr and Marletta, 1987). The chicken’s MHC (B complex) is known to influence the outcome of various diseases of viral, bacterial, and parasitic etiology (Bacon et al., 1981; Briles et al., 1983; Lamont et al., 1987; Lillehoj et al., 1989). The chromosomal location of the chicken iNOS gene is not yet known. However, the human iNOS gene is located on chromosome 17 (Chartrain et al., 1994), and is separate from the one that houses the MHC genes. It is interesting to speculate that one factor affecting geneticbased disease susceptibility and resistance differences in chickens may be due to differential iNOS activity. In fact, Taylor et al. (1992) have shown that dietary Larginine levels do influence Rous sarcoma-induced tumor growth in B-congenic chickens. The fact that our studies have clearly identified GB1 and GB2 as low responders and Cornell K-strain as the high responder chicken lines for iNOS expression and activity makes it possible to conduct disease challenge studies to test for biological correlation of iNOS activity with disease.

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HUSSAIN AND QURESHI nitrogen intermediate by macrophages of a uricotelic species. J. Leukocyte Biol. 50:49–56. Sung, Y. J., J. H. Hotchkiss, and R. R. Dietert, 1994. 2, 4-Diamino-6-hydroxypyrimidine, an inhibitor of GTP Cyclohydrolase I, suppresses nitric oxide production by chicken macrophages. Int. J. Immunopharmacol. 16: 101–108. Taylor, R. L., Jr., R. E. Austic, and R. R. Dietert, 1992. Dietary arginine influences Rous Sarcoma growth in a major histocompatibility B-complex progressor genotype. Proc. Soc. Exp. Biol. Med. 199:38–41. Unanue, E. R., and P. M. Allen, 1987. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 236:551–557. Xie, Q. W., H. J. Cho, J. Calaycay, R. A. Mumford, K. M. Swiderek, T. D. Lee, A. Ding, T. Troso, and C. Nathan, 1992. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256: 225–228. Xie, Q., Y. Kashiwabara, and C. Nathan, 1994. Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4705–4708.

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Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989. Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY. SAS Institute, 1989. SAS User’s Guide: Basics. Release 6.03 Edition. SAS Institute Inc., Cary, NC. Schmidt, H.H.H.W., and U. Walter, 1994. NO at work. Cell 78: 919–925. Stuehr, D. J., and M. A. Marletta, 1985. Mammalian nitrite biosynthesis: Mouse macrophage produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. USA. 82:7738–7742. Stuehr, D. J., and M. A. Marletta, 1987. Induction of nitrite/ nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-g. J. Immunol. 139:518–525. Stuehr, D. J., and C. F. Nathan, 1989. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169:1543–1555. Suk, K., S. D. Somers, and K. L. Erickson, 1993. Regulation of murine macrophage function by IL-4: IL-4 and IFN-g differentially regulate macrophage tumoricidal activation. Immunology 80:617–624. Sung, Y.-J., J. H. Hotchkiss, R. E. Austic, and R. R. Dietert, 1991. L-arginine dependent production of a reactive