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Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages
Veterinary Immunology and Immunopathology 90 (2002) 179–189
Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages Kevin A. Keane1, Gary L. Mason, James C. DeMartini* Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA Received 31 May 2002; received in revised form 17 September 2002; accepted 25 September 2002
Abstract Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Monocyte-derived macrophage; Nitric oxide; Ovine lentivirus; Reverse transcriptase
1. Introduction Ovine lentivirus (OvLV) also known as maedi-visna virus is the etiologic agent of lymphoid interstitial Abbreviations: Agu, aminoguanidine; HIV, human immunodeficiency virus; iNOS, inducible nitric oxide synthase; LIP, lymphoid interstitial pneumonia; MDMs, monocyte-derived macrophages; OvLV, ovine lentivirus; PBMCs, peripheral blood mononuclear cells; RT, reverse transcriptase * Corresponding author. Tel.: þ1-970-491-5410; fax: þ1-970-491-0603. E-mail addresses: [email protected] (K.A. Keane), [email protected] (J.C. DeMartini). 1 Present address: ICOS Corporation, 22021 20th AVE SE, Bothell, WA 98021, USA. Tel.: þ1-425-415-5109; fax: þ1-425-489-0356.
pneumonia (LIP) in sheep (DeMartini et al., 1993; Sigurdsson et al., 1952). LIP is a significant cause of morbidity and mortality in sheep (Narayan et al., 1993; Brodie et al., 1992) and also occurs in human and simian immunodeficiency virus infections (Jeena et al., 1998; Mankowski et al., 1998). Because in vivo infection and replication of OvLV occurs primarily in activated macrophages, and because activated macrophages are primary sources of nitric oxide, there may be a mechanistic link between lentivirus replication and nitric oxide. The purpose of this investigation was to examine the role of nitric oxide in early OvLV replication. Pulmonary nitric oxide is produced by alveolar macrophages and is an important molecule in the
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 2 4 5 - 3
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physiological and immunological activities of the lung (Trifilieff et al., 2000; Chung et al., 2001; Akaike, 2001; Duszyk and Radomski, 2000). Nitric oxide also plays a variety of roles in cellular activation (Lander, 1996; Bogdan, 2001) and has a controversial role in the replication of HIV (Blond et al., 2000; Mannick et al., 1999; Akaike and Maeda, 2000). There is considerable in vitro evidence that nitric oxide is capable of mitigating the functions of enzyme systems crucial to the replication of lentiviruses (Sehajpal et al., 1999; Colasanti et al., 1999; Sherry et al., 2000; Chen et al., 1999; Basu et al., 1999). However, it is unclear whether these pathways are identical in both cell populations that are infected by HIV, macrophages and T cells, as recent experiments indicate that nitric oxide modulates HIV replication in T cells differently than in macrophages (Jimenez et al., 2001). The experiments described herein use primary cells from the natural host to investigate early OvLV replication in macrophages without lymphocyte infection or depletion (Gorrell et al., 1992; Brodie et al., 1995; Blacklaws et al., 1995). There are many factors that influence the expression of the inducible nitric oxide synthase (iNOS) genes in macrophages and the subsequent production of nitric oxide. One consideration is that inter-species regulation of nitric oxide production seems to vary (Jungi et al., 1996; Lechner et al., 1999). Additionally, within a species, macrophages may alter nitric oxide production based on anatomical location (Wang et al., 1999), cellular differentiation (Jungi et al., 1996), or physiological demands (Zheng et al., 2000). Finally, there are a variety of infectious diseases in which nitric oxide production may be modified either directly by the pathogen (Bogdan et al., 1997; Mollace et al., 1993; Polazzi et al., 1999; Duranton et al., 1999) or indirectly from the immune response to the infectious agent (Blond et al., 1999; Jungi et al., 1997). In reality, it is likely a combination of pathogen and host influences which regulate nitric oxide (Mason et al., 1996; Laskin and Laskin, 1999). It was the objective of this investigation to clarify the role of nitric oxide in lentivirus replication. We observed treatments that inhibit nitric oxide production subsequently enhance viral replication. These observations suggest that nitric oxide is a potential mediator in the down regulation of viral replication after the initial infection and replicative burst observed in vivo.
2. Materials and methods 2.1. Monocyte-derived macrophages (MDMs) cultures MDM cultures generated from peripheral blood mononuclear cells (PBMCs) were isolated from ethylenediaminetetraacetic acid (EDTA) treated blood collected from a jugular vein and separated with Ficoll-Paque1 Plus (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s directions with slight modifications. The initial centrifuge step was altered to 670 g to more efficiently separate sheep PBMC from neutrophils. Cells were stained with trypan blue and viable cells were enumerated on a hemocytometer. All PBMC isolation procedures routinely had >95% viability. The PBMC were cultured using conditions similar to those previously described that induce monocytes to differentiate into macrophages (Narayan et al., 1983; Chebloune et al., 1996). The modified conditions included approximately 108 PBMC cultured in 30 ml of macrophage differentiation medium which consisted of macrophage-serum free medium, 10% heat-inactivated lamb serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from Gibco Life Technologies, Rockville, MD). The cultures were kept in Teflon1 coated flasks and placed in a 37 8C, 5% CO2 incubator for 1 week. The morphology of MDM cultures was examined by centrifuging 200,000 cells onto a glass slide and immunocytochemically staining for lysozyme as marker of macrophage differentiation. The primary antibody was rabbit anti-human lysozyme (Dako Corp., Glostrup, Denmark) and detection was accomplished with the Vectastain1 Elite ABC-Peroxidase kit and DAB substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Cells were lightly counterstained with hematoxylin and coverslipped. 2.2. In vitro OvLV infection After 1 week, the MDM cultures were split into two flasks. One flask was infected with OvLV strain 85/34 at an MOI of 3. This OvLV strain has been previously reported to produce lesions in vivo and replicate to high titers in vitro (Lairmore et al., 1987, 1988; Woodward et al., 1995). After infection the cells were
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washed once with macrophage differentiation medium and plated into either a 48 well plate or 8 well slide chamber at 50,000 cells per well. 2.3. Culture treatments Aminoguanidine is an amino acid analogue that binds to the active site of iNOS (Southan and Szabo, 1996) and was used to inhibit the production of nitric oxide. L-Arginine was added to the indicated cultures to compete with aminoguanidine for the iNOS active site. Bovine hemoglobin, which can trap free NO in culture supernatants was used to block the activity of NO (Blond et al., 2000). S-nitroso-acetylpenicillamine was used as an exogenous nitric oxide donor, while acetylpenicillamine was used as a negative control (Sehajpal et al., 1999). Final culture concentrations are indicated with the data. All culture treatment chemicals were acquired from Sigma Chemical Co. (St. Louis, MO). 2.4. Reverse transcriptase (RT) assay OvLV relative replication rates between treated cell cultures were deduced from the RT activity of the cell culture supernatant. The principle of this assay is that each virion that buds off an infected cell carries RT; the numbers of virions produced are proportional to the enzymatic activity of RT. To perform this assay, supernatants were collected from identically infected cell cultures with various treatments using the Lenti-RTTM Activity Assay (Cavidi Tech, Uppsala, Sweden) according to the manufacturer’s instructions with some modification. The supernatants were incubated in a microtiter plate coated with a RNA primer along with a solution containing bromine labeled nucleotides. After incubation and subsequent washing, the bromine labeled nucleotides incorporated were quantitated using alkaline phosphatase antibodies and the appropriate substrate which was subsequently measured at 405 nm on a MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Alterations to the procedure included testing all samples in quintuplicate and only testing non-diluted supernatants. Supernatants from uninfected MDM cultures were used as negative controls. Supernatants containing the original virus inoculum, collected from goat synovial membrane tissue cultures were used as positive controls.
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2.5. Measure of nitric oxide production Nitric oxide concentrations of the cell culture supernatant were deduced from the accumulation of aqueous nitrate and nitrite via the Griess reaction (Lander, 1996). In the Griess reaction, nitrite readily combines with N-(1-naphthyl) ethylenediamine dihydrochloride under acidic condition to form a colored azo dye which was quantitated with a microplate reader at 570 nm and compared to a standard curve made from sodium nitrite. This assay was performed using the nitrate/nitrite colorimetric assay kit (lactate dehydrogenase method) from Cayman Chemical Co. (Ann Arbor, MI) according to the manufacturer’s instructions. 2.6. Immunofluorescent staining Immunofluorescent staining was used to detect iNOS and OvLV antigen in the eight well tissue culture system. After the incubation period, the supernatants were removed, the cells were washed in saline followed by air-drying and fixation was done in cold acetone. All slides were stained at the same time by rehydrating the cells in saline, blocking with Protein Block Solution (Biogenex, San Ramon, CA), and quenching with 3% hydrogen peroxide. The primary antibodies were monoclonal 3F (Marcom et al., 1991) against OvLV capsid p25 and polyclonal against human iNOS (Zheng et al., 2000) (Santa Cruz Technology Inc., Santa Cruz, CA) which were used as previously reported. Calibration for immunofluorescent staining also included the omission of the primary antibody for determination of autofluorescence. Fluorescent labeling was accomplished using appropriate anti-isotype secondary antibodies conjugated to peroxidase and the Tyramide Signal AmplificationTM Plus Fluorescence System according to the manufacturer’s instructions (NEN Life Science Products, Boston, MA). Fluorescein was used to label viral antigen and cyanine 3 was used to label iNOS. Slides were coverslipped using Vectashield1 mounting medium containing DAPI (Vector Laboratories) and kept in the dark until viewing. 2.7. Image analysis for co-localization Quantitative image analysis involved capturing digital images (TIFF files) of fluorescent stained cells through an Olympus Microscope fitted with DAPI
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(610/675 nm), TRITC (557/590 nm), and FITC (500/ 525 nm) filters and a 3-CCD 1140 1520 detail Cool SNAP Camera (Roper Scientific GmbH, Bergkirchen, Germany). Five distinct images from each treatment group at each time point were captured using the 20 objective and analyzed with Metamorph software, Version 4.5 (Universal Imaging Corp., West Chester, PA) using the color thresholding, overlay, and co-localization tools. All fluorescent values were normalized for the cellular density of that particular image using the number of DAPI stained nuclei. The formula for this adjustment ¼ ðfluorescence area of imageÞ=ðtotal DAPI pixel area=10; 000Þ. Subsequently, the adjusted area of fluorescence corresponding to viral antigen was summed. Additionally, the area of fluorescence corresponding to iNOS production and co-localizing with viral antigen production was summed. 2.8. Statistical analyses All statistical analyses were performed with the Graphpad1 Instat software, Version 3.0 (San Diego, CA). Comparison of group mean data was analyzed for significance with a two-tailed, unpaired t-test with a Welch correction for means with different standard deviations. The dose response curve of RT activity to aminoguanidine treatment was assessed by a linear regression analysis. The aminoguanidine doses were log transformed and the virus only controls were assigned to the X-axis by one log value less than the lowest treatment dose. A linear correlation was calculated with the line forced through the Y-axis at the OD value of the sample with no drug treatment. The index value associating viral antigen and iNOS colocalization was established with a 2 2 contingency table using the presence or absence of iNOS on one axis compared to the presence or absence of viral antigen on the other axis. The chi square test was applied to the values. For all statistical tests, results were considered significant if the P value was <0.05.
3. Results 3.1. MDM cultures MDM cells consistently had excellent viability and were readily available for multiple simultaneous cell
Fig. 1. MDMs of sheep immunocytochemically stained for lysozyme as a marker of macrophage differentiation. (A) PBMC at the start of the culture. (B) MDM cells after 1 week in culture with a rare bi-nucleated cell. (C) MDM cells that have been infected with OvLV strain 85/34 for 3 days. Immunocytochemistry, lysozyme, horseradish peroxidase, hematoxylin counterstain bars ¼ 10 mm.
cultures. After the PBMC were cultured for 1 week, approximately 20% of the remaining cells were macrophages as assessed by lysozyme immunoreactivity (Fig. 1). The other 80% of the cells were washed away from the culture plates after the MDM cells were allowed to adhere. Lysozyme stain was not observed in any of the freshly isolated PBMC. The morphology of OvLV infected MDM cells was observed 48 h postinoculation (PI). The infected MDM cells appeared to have more vacuolation in the cytoplasm; however, the lysozyme immunoreactivity appeared similar to the non-infected MDM cells. Bi-nucleated MDM cells were observed in both infected and non-infected cultures. However, there were only 1–2 bi-nucleated cells per slide in the cytospin preparations from the noninfected MDM cells whereas there were 1–2 binucleated cells per high magnification field on the cytospin preparation from the OvLV inoculated culture.
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Fig. 2. RT activity of MDM cell culture supernatant at various times before and after inoculation. The pre-inoculum is concentrated OvLV strain 85/34 supernatant from goat synovial membrane cultures. The post-inoculum is the same supernatant after a 30 min incubation with the MDM cells at 37 8C in which there is a slight decrease in RT activity. Supernatants collected from uninfected MDM cells (negative), immediately after inoculation (0 h), and 24 h inoculation did not have RT activity. Only supernatant collected 48 h after virus inoculation had a significant ðP ¼ 0:01Þ increase in RT activity.
3.2. RT assay The original virus inoculum was tested for RT activity prior to inoculation and immediately after the 30 min incubation period. The source of the RT activity within the cultures was confirmed to be de novo virion from MDM cultures (Fig. 2). In this experiment, supernatants were collected prior to virus inoculation (negative), immediately after inoculation (time: 0 h), 24 h after inoculation, and 48 h after inoculation. The supernatant collected at 48 h PI and the positive controls were the only samples that tested positive (above the baseline) of RT activity. There was a slight decrease in the RT activity of the inoculum after the incubation period. Subsequent RT assays were performed 72 h after virus inoculation to maximize differences between treatment and control groups. Next, levels of RT activity were assessed when MDM cultures were treated with an exogenous source of nitric oxide (Fig. 3). S-nitroso-acetylpenicillamine (SNAP), which spontaneously donates NO in aqueous
Fig. 3. RT activity of MDM cell culture supernatant 72 h after in vitro OvLV inoculation with various concentrations of either 1 mM SNAP or 1 mM NAP. Cultures were also treated with 5 mM bovine hemoglobin (Hg) or 0.2 mM aminoguanidine (Agu). The addition of exogenous nitric oxide did not change the level of RT activity above background, but chemicals that either scavenged nitric oxide or blocked its production were associated with significant rises in RT activity.
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media was added to cultures at varying concentrations. Acetylpenicillamine (NAP) is a molecule without the nitric oxide donor molecule and was used as a negative control. Neither the exogenous nitric oxide donor nor the control molecule increased RT levels over background. The other half of this experiment was the addition of bovine hemoglobin to scavenge nitric oxide or aminoguanidine to block iNOS. There was a small rise with weak significance in the RT activity with the addition of hemoglobin to scavenge nitric oxide. However, there was large, strongly significant rise in RT activity when nitric oxide production is inhibited by aminoguanidine. A series of experiments were subsequently designed to confirm the observation that inhibition of nitric oxide production was functionally related to increases in OvLV replication. First an experiment was performed in which endogenous production of nitric oxide was blocked over a dose response curve and then RT activity was examined (Fig. 4). Aminoguanidine was again used as an amino acid analogue of arginine, which competes with L-arginine for the functional site of iNOS. As the aminoguanidine concentration increased there was a significant increase in the RT activity. As the aminoguanidine dose rose into the toxic dose range the RT activity began to plateau. A parallel experiment, with increasing doses of exogenous nitric oxide added to the culture was performed. In this experiment, a dose response curve was not observed. In order to confirm the mechanistic connection between aminoguanidine and viral replication, L-arginine was added to a culture along with aminoguanidine (Fig. 5). Under this culture condition, the Larginine will compete with the aminoguanidine for the active site of iNOS. In this experiment and similar to the previous experiment, there was a significant increase in the RT activity of the aminoguanidine treated culture. This increase was partially reversed by the addition of L-arginine to an aminoguanidine containing culture. The value of the RT activity in the aminoguanidine and L-arginine combination culture was statistically at median between the virus only and virus plus aminoguanidine culture. Concurrent measurements of nitric oxide concentrations in the supernatant were attempted in this experiment. These measurements were consistently below 10 mM and thus outside of the linear range of the plate reader.
Fig. 4. RT activity of MDM cell culture supernatant 72 h after OvLV inoculation with various treatments blocking or adding nitric oxide. (A) Aminoguanidine was added at various concentrations and resulted in a dose response line of RT activity that was significantly different than a slope of zero. (B) SNAP was added at various concentrations and resulted in a dose response line of RT activity that did not differ from a slope of zero. Line fitting was done with a first order polynomial equation. The zero doses were fitted on to the X-axis at value one log less than the lowest treatment dose.
3.3. Immunofluorescent staining of iNOS and OvLV antigen To examine the nitric oxide–OvLV replication relationship by an alternate method, the production of
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Fig. 5. RT activity and nitrite accumulation in MDM cell culture supernatant 72 h after OvLV inoculation. The cultures were treated with either 0.2 mM aminoguanidine (Agu) or 1.0 mM L-arginine (LA). Left Y-axis: there was a significant ðP < 0:01Þ increase in the RT activity of the aminoguanidine treated culture. The increased RT activity was partially reversed by the addition of L-arginine to an aminoguanidine containing culture (26% reduction in RT activity, P ¼ 0:02). Right Y-axis: nitric oxide was measured in the supernatant by reducing nitrate to nitrite with subsequent quantification of nitrite using the Griess reaction in which samples are compared to a standard curve of sodium nitrite was below the linear range of the assay.
iNOS and OvLV antigen were measured in MDM cells by immunofluorescent co-localization technique. Antibodies against OvLV capsid and iNOS were first tested for specific staining using chromagenic detection of OvLV infected MDM cytospin preparations (Fig. 6). Once the specific staining was confirmed, OvLV inoculated MDM cultures were examined over time for the percentage of cells producing virus antigen and evidence of co-localization with iNOS Table 1 Analysis of co-localization of OvLV and iNOS using a chi square test of a 2 2 contingency tablea
iNOS positive cell iNOS negative cells Total a
OvLV positive cells
OvLV negative cells
Total
400 (6%) 1514 (23%) 1914 (29%)
798 (12%) 3853 (59%) 4651 (71%)
1198 (18%) 5367 (82%) 6565 (100%)
The cell counts in each category were determined at 64 h PI when the cultures were treated with aminoguanidine. The row/ column association has a two-sided P value of 0.0004. The relative risk of OvLV positive and iNOS positive cells co-localizing is 1.184 (95% confidence interval ¼ 1:081–1.296).
(Fig. 6). The cumulative level of viral antigen positive cells exceeded background levels only in the latter portion of the experiment (64 and 72 h PI). Additionally, a rise above background in viral antigen associated fluorescence was only observed in the aminoguanidine treated cultures. Fluorescent co-localization was analyzed by the chi square test (Table 1). This test was only applied to time points PI in which the total viral antigen fluorescence was distinguished above autofluorescence levels. At 64 h PI, the relative risk of co-localization was calculated to be 1.184 (95% confidence interval ¼ 1:0811:296, P ¼ 0:0004).
4. Discussion This investigation provides two lines of evidence that nitric oxide negatively regulates lentivirus replication in MDMs: first, the addition of aminoguanidine to cultured macrophages infected with OvLV significantly increases the RT activity of the culture supernatant. Second, in a separate experiment, the addition of aminoguanidine to a similar culture system increased the production of OvLV capsid protein within
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Fig. 6. Relative co-localization of OvLV antigen (capsid) and iNOS in monocyte-derived macrophages (MDM). (A) L-Arginine treated and (B) immunofluorescent images of OvLV capsid (FITC-green) and iNOS (Cy3-red) in aminoguanidine treated MDM cell cultures at 72 h PI. All nuclei stained blue (DAPI). The white arrows point to multinucleated cells that are strongly positive for OvLV antigen expression. (C) Immunofluorescent co-localization of OvLV capsid antigen and iNOS in MDM cell cultures treated with aminoguanidine (AG) and L-arginine (LA). The bar graph represents the sum fluorescent area of OvLV capsid producing cells adjusted for cellular density (see Section 2 for formula). The black portions of the bars represent the area of fluorescence at each point that co-localized with iNOS production in the same cell. OvLV capsid expression was significantly ðP ¼ 0:01Þ above autofluorescence levels at 64 and 72 h PI only in the aminoguanidine treated culture.
the cytoplasm of infected macrophages. These two observations in the virus-infected cultures in which the production of nitric oxide is inhibited, suggest that the presence of nitric oxide may be an innate mechanism
of down regulating lentiviral replication following OvLVs initial replicative burst. RT activity was chosen as an indicator for viral replication as this enzyme is packaged within each
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virion during assembly. Thus, the quantity of RT activity in supernatant is presumed to be proportional to the number of virions in the supernatant. Since each well contained similar numbers of cells, the difference in RT activity was attributed to the treatments. The RT kit used was designed for quantitative analyses. Furthermore, RT activity was not detected sooner than 48 h after inoculation. Thus, the measured RT activity was from de novo virus production rather than carryover from the original inoculum. The interpretation that the presence of aminoguanidine increased RT activity is reinforced by a significant dose response curve. Finally, the addition of excess L-arginine, which competes directly with aminoguanidine for the active site of iNOS, partially counteracted the effects of the aminoguanidine. The experiments involving OvLV antigen production were performed to confirm that the level of RT activity was truly indicative of virus replication and refute the possibility that treatments were merely altering RT in vitro activity and not directly affecting virus replication. This alteration in RT activity could include direct interference, such as L-arginine inhibiting RT activity, or indirect interference with nitric oxide, reducing RT activity but not necessarily altering virus replication. The presence of virus antigen within MDM cell cytoplasm under the circumstances of this experiment is independent of RT activity. That is to say that although RT was necessary to initially create a provirus containing cell, that step was prior to any treatment and should be constant between aminoguanidine and L-arginine treated cultures. Conversely, the antigen measured in this experiment is viral capsid. The virus production was based on the number of cells containing virus capsid and not the activity of an enzyme. Thus, both the experiments involving measurement of RT activity and the experiment quantifying the number of OvLV antigen positive cells indicate that inhibition of nitric oxide synthase creates an environment which enhances OvLV replication. It was not entirely clear why the addition of exogenous nitric oxide did not further lower the RT activity of the treated cultures. One possible explanation is that a floor effect had been reached with the endogenous production of nitric oxide and addition of exogenous nitric oxide was not going to alter that lower RT activity. Alternatively, the half-life of the exogenously added nitric oxide (estimated by the
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manufacturer to be only a few minutes) might have been an insufficient exposure time to reduce viral replication as reflected in RT activity. As mentioned in Section 1, the regulation of iNOS in sheep has been difficult to define due to the low levels of nitrate accumulation in sheep cell cultures (Bogdan et al., 1997). This investigation also had difficulty in establishing functional relationships based on nitrate accumulation since the quantities of nitrite in the supernatants were outside the linear range of the Griess reaction to accurately measure changes. It is possible that measuring the levels of intracellular nitric oxide might resolve this investigative obstacle. The experimental design used herein does not address which step of viral replication may be altered by nitric oxide. Nitric oxide may influence viral replication at translation, transcription, virion packaging, or budding from the host cell. It is also possible that nitric oxide may act in concert with tumor necrosis factor-a (TNF-a) to influence viral replication since TNF-a is a potent mediator of nitric oxide. A further possibility exists that aminoguanidine may influence the nitric oxide to TNF-a ratio; thus indirectly altering viral replication (Rashid et al., 2001; Bacsi et al., 2001). Experiments addressing these questions would be a logical progression of this investigation. The results in the present investigation demonstrate the value of using sheep MDM cells to investigate mechanisms that affect lentivirus infection and replication. MDM cells were chosen over other cell populations such as alveolar macrophages because of their ease of acquisition and hardiness in culture. Although not directly measured in this investigation, it was presumed that the cell cultures were largely depleted of lymphocytes during the washing and media change steps. The MDM cells were strongly adhered to plastic culture surfaces whereas the lymphocytes are less so. Thus, intra-culture cytotoxicity from activated lymphocytes was presumed to not occur in this investigation. In conclusion, we believe that nitric oxide is part of the innate host response to lentivirus infection that down regulates OvLV replication in macrophages. The challenge that remains is to integrate our understanding of this innate response with the adaptive response of the host’s immune system to further define host– virus relationship.
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Acknowledgements This work was supported by NHLBI-NIH grant K08 HL 03792 to Dr. Kevin Keane.
References Akaike, T., 2001. Role of free radicals in viral pathogenesis and mutation. Rev. Med. Virol. 11, 87–101. Akaike, T., Maeda, H., 2000. Nitric oxide and virus infection. Immunology 101, 300–308. Bacsi, A., Csoma, E., Beck, Z., Andirko, I., Konya, J., Gergely, L., Toth, F.D., 2001. Induction of HIV-1 replication in latently infected syncytiotrophoblast cells by contact with placental macrophages: role of interleukin-6 and tumor necrosis factoralpha. J. Interferon Cytokine Res. 21, 1079–1088. Basu, A., Sehajpal, P.K., Ogiste, J.S., Lander, H.M., 1999. Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species. Antioxide Redox Signal 1, 105–112. Blacklaws, B.A., Bird, P., Allen, D., Roy, D.J., MacLennan, C.M., Hopkins, J., Sargan, D.R., McConnell, I., 1995. Initial lentivirus– host interactions within lymph nodes: a study of maedi-visna virus infection in sheep. J. Virol. 69 (3), 1400–1407. Blond, D., Cheret, A., Raoul, H., Le Grand, R., Caufour, P., Theodoro, F., Dormont, D., 1999. Nitric oxide synthesis during acute SIV mac251 infection of macaques. Res. Virol. 149, 75–86. Blond, D., Raoul, H., Le Grand, R., Dormont, D., 2000. Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages. J. Virol. 74, 8904–8912. Bogdan, C., 2001. Nitric oxide and the regulation of gene expression. Trends Cell Biol. 11, 66–75. Bogdan, J.R., Newlands-Monteith, C.F., Ellis, J.A., 1997. Nitric oxide production following in vitro stimulation of ovine pulmonary alveolar macrophages. Vet. Immunol. Immunopathol. 56, 299–310. Brodie, S.J., Marcom, K.A., Pearson, L.D., Anderson, B.C., de la Concha-Bermejillo, A., Ellis, J.A., DeMartini, J.C., 1992. Effects of virus load in the pathogenesis of lentivirus-induced lymphoid interstitial pneumonia. J. Infect. Dis. 166, 531–541. Brodie, S.J., Pearson, L.D., Zink, M.C., Bickle, H.M., Anderson, B.C., Marcom, K.A., DeMartini, J.C., 1995. Ovine lentivirus expression and disease virus replication, but not entry, is restricted to macrophages of specific tissues. Am. J. Pathol. 146, 250–263. Chebloune, Y., Karr, B., Sheffer, D., Leung, K., Narayan, O., 1996. Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep. J. Gen. Virol. 77, 2037–2051. Chen, F., Lu, Y., Castranova, V., Rojanasakul, Y., Miyahara, K., Shizuta, Y., Vallyathan, V., Shi, X., Demers, L.M., 1999. Nitric oxide inhibits HIV tat-induced NF-kappaB activation. Am. J. Pathol. 155, 275–284.
Chung, H.T., Pae, H.O., Choi, B.M., Billiar, T.R., Kim, Y.M., 2001. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun. 282, 1075–1079. Colasanti, M., Persichini, T., Venturini, G., Ascenzi, P., 1999. Snitrosylation of viral proteins: molecular bases for antiviral effect of nitric oxide. IUBMB Life 48, 25–31. DeMartini, J.C., Brodie, S.J., de la Concha-Bermejillo, A., Ellis, J.A., Lairmore, M.D., 1993. Pathogenesis of lymphoid interstitial pneumonia in natural and experimental ovine lentivirus infection. Clinical Infect. Dis. 17, 236–242. Duranton, C., Dorchies, P., Grand, S., Lesure, C., Oswald, I.P., 1999. Changing reactivity of caprine and ovine mononuclear phagocytes throughout part of the life cycle of Oestrus ovis: assessment through spontaneous and inductible NO production. Vet. Res. 30, 371–376. Duszyk, M., Radomski, M.W., 2000. The role of nitric oxide in the regulation of ion channels in airway epithelium: implications for diseases of the lung. Free Rad. Res. 33, 449–459. Gorrell, M.D., Brandon, M.R., Sheffer, D., Adams, R.J., Narayan, O., 1992. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J. Virol. 66, 2679–2688. Jeena, P.M., Coovadia, H.M., Thula, S.A., Blythe, D., Buckels, N.J., Chetty, R., 1998. Persistent and chronic lung disease in HIV-1 infected and uninfected African children. AIDS 12, 1185–1193. Jimenez, J.L., Gonzalez-Nicolas, J., Alvarez, S., Fresno, M., Munoz-Fernandez, M.A., 2001. Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide. J. Virol. 75, 4655–4663. Jungi, T.W., Thony, M., Brcic, M., Adler, B., Pauli, U., Peterhans, E., 1996. Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent. Immunobiology 195, 385–400. Jungi, T.W., Pfister, H., Sager, H., Fatzer, R., Vandevelde, M., Zurbriggen, A., 1997. Comparison of inducible nitric oxide synthase expression in the brains of listeria monocytogenesinfected cattle, sheep, and goats and in macrophages stimulated in vitro. Infect. Immun. 65, 5279–5288. Lairmore, M.D., Akita, G.Y., Russell, H.R., DeMartini, J.C., 1987. Replication and cytopathic effects of ovine lentivirus strains in alveolar macrophages correlate with in vivo pathogenicity. J. Virol. 61, 4038–4042. Lairmore, M.D., Poulson, J.M., Adducci, T.A., DeMartini, J.C., 1988. Lentivirus-induced lymphoproliferative disease. Comparative pathogenicity of distinct ovine lentivirus strains. Am. J. Pathol. 130, 80–89. Lander, H.M., 1996. Cellular activation mediated by nitric oxide. Methods 10, 15–20. Laskin, J.D., Laskin, D.L., 1999. Cellular and Molecular Biology of Nitric Oxide. Marcel Dekker, New York. Lechner, F., Schutte, A., Von Bodungen, U., Bertoni, G., Pfister, H., Jungi, T.W., Peterhans, E., 1999. Inducible nitric oxide synthase is expressed in joints of goats in the late stage of infection with caprine arthritis encephalitis virus. Clin. Exp. Immunol. 117, 70–75. Mankowski, J.L., Carter, D.L., Spelman, J.P., Nealen, M.L., Maughan, K.R., Kirstein, L.M., Didier, P.J., Adams, R.J.,
K.A. Keane et al. / Veterinary Immunology and Immunopathology 90 (2002) 179–189 Murphey-Corb, M., Zink, M.C., 1998. Pathogenesis of simian immunodeficiency virus pneumonia: an immunopathological response to virus. Am. J. Pathol. 153, 1123–1130. Mannick, J.B., Stamler, J.S., Teng, E., Simpson, N., Lawrence, J., Jordan, J., Finberg, R.W., 1999. Nitric oxide modulates HIV-1 replication. J. Acq. Immun. Def. Syn. 22, 1–9. Marcom, K.A., Pearson, L.D., Chung, C.S., Poulson, J.M., DeMartini, J.C., 1991. Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates. J. Clin. Microbiol. 29, 1472–1479. Mason, G.L., Yang, Z., Olchowy, T.W., Jian, Z., Bochsler, P.N., 1996. Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages. Vet. Immunol. Immunopathol. 53, 15–27. Mollace, V., Colasanti, M., Persichini, T., Bagetta, G., Lauro, G.M., Nistico, G., 1993. HIV gp120 glycoprotein stimulates the inducible isoform of no synthase in human cultured astrocytoma cells. Biochem. Biophys. Res. Commun. 194, 439–445. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D.E., Clements, J.E., 1983. Activation of caprine arthritis-encephalitis virus expression during maturation of monocytes to macrophages. Infect. Immun. 41, 67–73. Narayan, O., Zink, M.C., Gorrell, M., Crane, S., Huso, D., Jolly, P., Saltarelli, M., Adams, R.B., Clements, J.E., 1993. The lentiviruses of sheep and goats. In: Levy, J.A. (Ed.), The Retroviridae. Plenum Press, New York, pp. 229–255. Polazzi, E., Levi, G., Minghetti, L., 1999. Human immunodeficiency virus type 1 Tat protein stimulates inducible nitric oxide synthase expression and nitric oxide production in microglial cultures. J. Neuropathol. Exp. Neurol. 58, 825–831. Rashid, G., Luzon, A.A., Korzets, Z., Klein, O., Zeltzer, E., Bernheim, J., 2001. The effect of advanced glycation end-
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products and aminoguanidine on TNFalpha production by rat peritoneal macrophages. Perit. Dial. Int. 21, 122–129. Sehajpal, P.K., Basu, A., Ogiste, J.S., Lander, H.M., 1999. Reversible S-nitrosation and inhibition of HIV-1 protease. Biochemistry 38, 13407–13413. Sherry, B., Schmidtmayerova, H., Zybarth, G., Dubrovsky, L., Raabe, T., Bukrinsky, M., 2000. Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response. Mol. Med. 6, 542–549. Sigurdsson, B., Grimsson, H., Pa´ lsson, P.A., 1952. Maedi, a chronic, progressive infection of sheep’s lungs. J. Infect. Dis. 90, 233–241. Southan, G.J., Szabo, C., 1996. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 51, 383–394. Trifilieff, A., Fujitani, Y., Mentz, F., Dugas, B., Fuentes, M., Bertrand, C., 2000. Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression. J. Immunol. 165, 1526–1533. Wang, M.J., Jeng, K.C., Shih, P.C., 1999. Differential expression of inducible nitric oxide synthase gene by alveolar and peritoneal macrophages in lipopolysaccharide-hyporesponsive C3H/HeJ mice. Immunology 98, 497–503. Woodward, T.M., Carlson, J.O., de la Concha-Bermejillo, A., DeMartini, J.C., 1995. Biological and genetic changes in ovine lentivirus strains following passage in isogeneic twin lambs. J. Acq. Immun. Def. Syn. 8, 124–133. Zheng, J., Li, Y., Weiss, A.R., Bird, I.M., Magness, R.R., 2000. Expression of endothelial and inducible nitric oxide synthases and nitric oxide production in ovine placental and uterine tissues during late pregnancy. Placenta 21, 516–524.
Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages Kevin A. Keane1, Gary L. Mason, James C. DeMartini* Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA Received 31 May 2002; received in revised form 17 September 2002; accepted 25 September 2002
Abstract Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Monocyte-derived macrophage; Nitric oxide; Ovine lentivirus; Reverse transcriptase
1. Introduction Ovine lentivirus (OvLV) also known as maedi-visna virus is the etiologic agent of lymphoid interstitial Abbreviations: Agu, aminoguanidine; HIV, human immunodeficiency virus; iNOS, inducible nitric oxide synthase; LIP, lymphoid interstitial pneumonia; MDMs, monocyte-derived macrophages; OvLV, ovine lentivirus; PBMCs, peripheral blood mononuclear cells; RT, reverse transcriptase * Corresponding author. Tel.: þ1-970-491-5410; fax: þ1-970-491-0603. E-mail addresses: [email protected] (K.A. Keane), [email protected] (J.C. DeMartini). 1 Present address: ICOS Corporation, 22021 20th AVE SE, Bothell, WA 98021, USA. Tel.: þ1-425-415-5109; fax: þ1-425-489-0356.
pneumonia (LIP) in sheep (DeMartini et al., 1993; Sigurdsson et al., 1952). LIP is a significant cause of morbidity and mortality in sheep (Narayan et al., 1993; Brodie et al., 1992) and also occurs in human and simian immunodeficiency virus infections (Jeena et al., 1998; Mankowski et al., 1998). Because in vivo infection and replication of OvLV occurs primarily in activated macrophages, and because activated macrophages are primary sources of nitric oxide, there may be a mechanistic link between lentivirus replication and nitric oxide. The purpose of this investigation was to examine the role of nitric oxide in early OvLV replication. Pulmonary nitric oxide is produced by alveolar macrophages and is an important molecule in the
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 2 4 5 - 3
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physiological and immunological activities of the lung (Trifilieff et al., 2000; Chung et al., 2001; Akaike, 2001; Duszyk and Radomski, 2000). Nitric oxide also plays a variety of roles in cellular activation (Lander, 1996; Bogdan, 2001) and has a controversial role in the replication of HIV (Blond et al., 2000; Mannick et al., 1999; Akaike and Maeda, 2000). There is considerable in vitro evidence that nitric oxide is capable of mitigating the functions of enzyme systems crucial to the replication of lentiviruses (Sehajpal et al., 1999; Colasanti et al., 1999; Sherry et al., 2000; Chen et al., 1999; Basu et al., 1999). However, it is unclear whether these pathways are identical in both cell populations that are infected by HIV, macrophages and T cells, as recent experiments indicate that nitric oxide modulates HIV replication in T cells differently than in macrophages (Jimenez et al., 2001). The experiments described herein use primary cells from the natural host to investigate early OvLV replication in macrophages without lymphocyte infection or depletion (Gorrell et al., 1992; Brodie et al., 1995; Blacklaws et al., 1995). There are many factors that influence the expression of the inducible nitric oxide synthase (iNOS) genes in macrophages and the subsequent production of nitric oxide. One consideration is that inter-species regulation of nitric oxide production seems to vary (Jungi et al., 1996; Lechner et al., 1999). Additionally, within a species, macrophages may alter nitric oxide production based on anatomical location (Wang et al., 1999), cellular differentiation (Jungi et al., 1996), or physiological demands (Zheng et al., 2000). Finally, there are a variety of infectious diseases in which nitric oxide production may be modified either directly by the pathogen (Bogdan et al., 1997; Mollace et al., 1993; Polazzi et al., 1999; Duranton et al., 1999) or indirectly from the immune response to the infectious agent (Blond et al., 1999; Jungi et al., 1997). In reality, it is likely a combination of pathogen and host influences which regulate nitric oxide (Mason et al., 1996; Laskin and Laskin, 1999). It was the objective of this investigation to clarify the role of nitric oxide in lentivirus replication. We observed treatments that inhibit nitric oxide production subsequently enhance viral replication. These observations suggest that nitric oxide is a potential mediator in the down regulation of viral replication after the initial infection and replicative burst observed in vivo.
2. Materials and methods 2.1. Monocyte-derived macrophages (MDMs) cultures MDM cultures generated from peripheral blood mononuclear cells (PBMCs) were isolated from ethylenediaminetetraacetic acid (EDTA) treated blood collected from a jugular vein and separated with Ficoll-Paque1 Plus (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s directions with slight modifications. The initial centrifuge step was altered to 670 g to more efficiently separate sheep PBMC from neutrophils. Cells were stained with trypan blue and viable cells were enumerated on a hemocytometer. All PBMC isolation procedures routinely had >95% viability. The PBMC were cultured using conditions similar to those previously described that induce monocytes to differentiate into macrophages (Narayan et al., 1983; Chebloune et al., 1996). The modified conditions included approximately 108 PBMC cultured in 30 ml of macrophage differentiation medium which consisted of macrophage-serum free medium, 10% heat-inactivated lamb serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from Gibco Life Technologies, Rockville, MD). The cultures were kept in Teflon1 coated flasks and placed in a 37 8C, 5% CO2 incubator for 1 week. The morphology of MDM cultures was examined by centrifuging 200,000 cells onto a glass slide and immunocytochemically staining for lysozyme as marker of macrophage differentiation. The primary antibody was rabbit anti-human lysozyme (Dako Corp., Glostrup, Denmark) and detection was accomplished with the Vectastain1 Elite ABC-Peroxidase kit and DAB substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Cells were lightly counterstained with hematoxylin and coverslipped. 2.2. In vitro OvLV infection After 1 week, the MDM cultures were split into two flasks. One flask was infected with OvLV strain 85/34 at an MOI of 3. This OvLV strain has been previously reported to produce lesions in vivo and replicate to high titers in vitro (Lairmore et al., 1987, 1988; Woodward et al., 1995). After infection the cells were
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washed once with macrophage differentiation medium and plated into either a 48 well plate or 8 well slide chamber at 50,000 cells per well. 2.3. Culture treatments Aminoguanidine is an amino acid analogue that binds to the active site of iNOS (Southan and Szabo, 1996) and was used to inhibit the production of nitric oxide. L-Arginine was added to the indicated cultures to compete with aminoguanidine for the iNOS active site. Bovine hemoglobin, which can trap free NO in culture supernatants was used to block the activity of NO (Blond et al., 2000). S-nitroso-acetylpenicillamine was used as an exogenous nitric oxide donor, while acetylpenicillamine was used as a negative control (Sehajpal et al., 1999). Final culture concentrations are indicated with the data. All culture treatment chemicals were acquired from Sigma Chemical Co. (St. Louis, MO). 2.4. Reverse transcriptase (RT) assay OvLV relative replication rates between treated cell cultures were deduced from the RT activity of the cell culture supernatant. The principle of this assay is that each virion that buds off an infected cell carries RT; the numbers of virions produced are proportional to the enzymatic activity of RT. To perform this assay, supernatants were collected from identically infected cell cultures with various treatments using the Lenti-RTTM Activity Assay (Cavidi Tech, Uppsala, Sweden) according to the manufacturer’s instructions with some modification. The supernatants were incubated in a microtiter plate coated with a RNA primer along with a solution containing bromine labeled nucleotides. After incubation and subsequent washing, the bromine labeled nucleotides incorporated were quantitated using alkaline phosphatase antibodies and the appropriate substrate which was subsequently measured at 405 nm on a MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Alterations to the procedure included testing all samples in quintuplicate and only testing non-diluted supernatants. Supernatants from uninfected MDM cultures were used as negative controls. Supernatants containing the original virus inoculum, collected from goat synovial membrane tissue cultures were used as positive controls.
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2.5. Measure of nitric oxide production Nitric oxide concentrations of the cell culture supernatant were deduced from the accumulation of aqueous nitrate and nitrite via the Griess reaction (Lander, 1996). In the Griess reaction, nitrite readily combines with N-(1-naphthyl) ethylenediamine dihydrochloride under acidic condition to form a colored azo dye which was quantitated with a microplate reader at 570 nm and compared to a standard curve made from sodium nitrite. This assay was performed using the nitrate/nitrite colorimetric assay kit (lactate dehydrogenase method) from Cayman Chemical Co. (Ann Arbor, MI) according to the manufacturer’s instructions. 2.6. Immunofluorescent staining Immunofluorescent staining was used to detect iNOS and OvLV antigen in the eight well tissue culture system. After the incubation period, the supernatants were removed, the cells were washed in saline followed by air-drying and fixation was done in cold acetone. All slides were stained at the same time by rehydrating the cells in saline, blocking with Protein Block Solution (Biogenex, San Ramon, CA), and quenching with 3% hydrogen peroxide. The primary antibodies were monoclonal 3F (Marcom et al., 1991) against OvLV capsid p25 and polyclonal against human iNOS (Zheng et al., 2000) (Santa Cruz Technology Inc., Santa Cruz, CA) which were used as previously reported. Calibration for immunofluorescent staining also included the omission of the primary antibody for determination of autofluorescence. Fluorescent labeling was accomplished using appropriate anti-isotype secondary antibodies conjugated to peroxidase and the Tyramide Signal AmplificationTM Plus Fluorescence System according to the manufacturer’s instructions (NEN Life Science Products, Boston, MA). Fluorescein was used to label viral antigen and cyanine 3 was used to label iNOS. Slides were coverslipped using Vectashield1 mounting medium containing DAPI (Vector Laboratories) and kept in the dark until viewing. 2.7. Image analysis for co-localization Quantitative image analysis involved capturing digital images (TIFF files) of fluorescent stained cells through an Olympus Microscope fitted with DAPI
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(610/675 nm), TRITC (557/590 nm), and FITC (500/ 525 nm) filters and a 3-CCD 1140 1520 detail Cool SNAP Camera (Roper Scientific GmbH, Bergkirchen, Germany). Five distinct images from each treatment group at each time point were captured using the 20 objective and analyzed with Metamorph software, Version 4.5 (Universal Imaging Corp., West Chester, PA) using the color thresholding, overlay, and co-localization tools. All fluorescent values were normalized for the cellular density of that particular image using the number of DAPI stained nuclei. The formula for this adjustment ¼ ðfluorescence area of imageÞ=ðtotal DAPI pixel area=10; 000Þ. Subsequently, the adjusted area of fluorescence corresponding to viral antigen was summed. Additionally, the area of fluorescence corresponding to iNOS production and co-localizing with viral antigen production was summed. 2.8. Statistical analyses All statistical analyses were performed with the Graphpad1 Instat software, Version 3.0 (San Diego, CA). Comparison of group mean data was analyzed for significance with a two-tailed, unpaired t-test with a Welch correction for means with different standard deviations. The dose response curve of RT activity to aminoguanidine treatment was assessed by a linear regression analysis. The aminoguanidine doses were log transformed and the virus only controls were assigned to the X-axis by one log value less than the lowest treatment dose. A linear correlation was calculated with the line forced through the Y-axis at the OD value of the sample with no drug treatment. The index value associating viral antigen and iNOS colocalization was established with a 2 2 contingency table using the presence or absence of iNOS on one axis compared to the presence or absence of viral antigen on the other axis. The chi square test was applied to the values. For all statistical tests, results were considered significant if the P value was <0.05.
3. Results 3.1. MDM cultures MDM cells consistently had excellent viability and were readily available for multiple simultaneous cell
Fig. 1. MDMs of sheep immunocytochemically stained for lysozyme as a marker of macrophage differentiation. (A) PBMC at the start of the culture. (B) MDM cells after 1 week in culture with a rare bi-nucleated cell. (C) MDM cells that have been infected with OvLV strain 85/34 for 3 days. Immunocytochemistry, lysozyme, horseradish peroxidase, hematoxylin counterstain bars ¼ 10 mm.
cultures. After the PBMC were cultured for 1 week, approximately 20% of the remaining cells were macrophages as assessed by lysozyme immunoreactivity (Fig. 1). The other 80% of the cells were washed away from the culture plates after the MDM cells were allowed to adhere. Lysozyme stain was not observed in any of the freshly isolated PBMC. The morphology of OvLV infected MDM cells was observed 48 h postinoculation (PI). The infected MDM cells appeared to have more vacuolation in the cytoplasm; however, the lysozyme immunoreactivity appeared similar to the non-infected MDM cells. Bi-nucleated MDM cells were observed in both infected and non-infected cultures. However, there were only 1–2 bi-nucleated cells per slide in the cytospin preparations from the noninfected MDM cells whereas there were 1–2 binucleated cells per high magnification field on the cytospin preparation from the OvLV inoculated culture.
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Fig. 2. RT activity of MDM cell culture supernatant at various times before and after inoculation. The pre-inoculum is concentrated OvLV strain 85/34 supernatant from goat synovial membrane cultures. The post-inoculum is the same supernatant after a 30 min incubation with the MDM cells at 37 8C in which there is a slight decrease in RT activity. Supernatants collected from uninfected MDM cells (negative), immediately after inoculation (0 h), and 24 h inoculation did not have RT activity. Only supernatant collected 48 h after virus inoculation had a significant ðP ¼ 0:01Þ increase in RT activity.
3.2. RT assay The original virus inoculum was tested for RT activity prior to inoculation and immediately after the 30 min incubation period. The source of the RT activity within the cultures was confirmed to be de novo virion from MDM cultures (Fig. 2). In this experiment, supernatants were collected prior to virus inoculation (negative), immediately after inoculation (time: 0 h), 24 h after inoculation, and 48 h after inoculation. The supernatant collected at 48 h PI and the positive controls were the only samples that tested positive (above the baseline) of RT activity. There was a slight decrease in the RT activity of the inoculum after the incubation period. Subsequent RT assays were performed 72 h after virus inoculation to maximize differences between treatment and control groups. Next, levels of RT activity were assessed when MDM cultures were treated with an exogenous source of nitric oxide (Fig. 3). S-nitroso-acetylpenicillamine (SNAP), which spontaneously donates NO in aqueous
Fig. 3. RT activity of MDM cell culture supernatant 72 h after in vitro OvLV inoculation with various concentrations of either 1 mM SNAP or 1 mM NAP. Cultures were also treated with 5 mM bovine hemoglobin (Hg) or 0.2 mM aminoguanidine (Agu). The addition of exogenous nitric oxide did not change the level of RT activity above background, but chemicals that either scavenged nitric oxide or blocked its production were associated with significant rises in RT activity.
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media was added to cultures at varying concentrations. Acetylpenicillamine (NAP) is a molecule without the nitric oxide donor molecule and was used as a negative control. Neither the exogenous nitric oxide donor nor the control molecule increased RT levels over background. The other half of this experiment was the addition of bovine hemoglobin to scavenge nitric oxide or aminoguanidine to block iNOS. There was a small rise with weak significance in the RT activity with the addition of hemoglobin to scavenge nitric oxide. However, there was large, strongly significant rise in RT activity when nitric oxide production is inhibited by aminoguanidine. A series of experiments were subsequently designed to confirm the observation that inhibition of nitric oxide production was functionally related to increases in OvLV replication. First an experiment was performed in which endogenous production of nitric oxide was blocked over a dose response curve and then RT activity was examined (Fig. 4). Aminoguanidine was again used as an amino acid analogue of arginine, which competes with L-arginine for the functional site of iNOS. As the aminoguanidine concentration increased there was a significant increase in the RT activity. As the aminoguanidine dose rose into the toxic dose range the RT activity began to plateau. A parallel experiment, with increasing doses of exogenous nitric oxide added to the culture was performed. In this experiment, a dose response curve was not observed. In order to confirm the mechanistic connection between aminoguanidine and viral replication, L-arginine was added to a culture along with aminoguanidine (Fig. 5). Under this culture condition, the Larginine will compete with the aminoguanidine for the active site of iNOS. In this experiment and similar to the previous experiment, there was a significant increase in the RT activity of the aminoguanidine treated culture. This increase was partially reversed by the addition of L-arginine to an aminoguanidine containing culture. The value of the RT activity in the aminoguanidine and L-arginine combination culture was statistically at median between the virus only and virus plus aminoguanidine culture. Concurrent measurements of nitric oxide concentrations in the supernatant were attempted in this experiment. These measurements were consistently below 10 mM and thus outside of the linear range of the plate reader.
Fig. 4. RT activity of MDM cell culture supernatant 72 h after OvLV inoculation with various treatments blocking or adding nitric oxide. (A) Aminoguanidine was added at various concentrations and resulted in a dose response line of RT activity that was significantly different than a slope of zero. (B) SNAP was added at various concentrations and resulted in a dose response line of RT activity that did not differ from a slope of zero. Line fitting was done with a first order polynomial equation. The zero doses were fitted on to the X-axis at value one log less than the lowest treatment dose.
3.3. Immunofluorescent staining of iNOS and OvLV antigen To examine the nitric oxide–OvLV replication relationship by an alternate method, the production of
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Fig. 5. RT activity and nitrite accumulation in MDM cell culture supernatant 72 h after OvLV inoculation. The cultures were treated with either 0.2 mM aminoguanidine (Agu) or 1.0 mM L-arginine (LA). Left Y-axis: there was a significant ðP < 0:01Þ increase in the RT activity of the aminoguanidine treated culture. The increased RT activity was partially reversed by the addition of L-arginine to an aminoguanidine containing culture (26% reduction in RT activity, P ¼ 0:02). Right Y-axis: nitric oxide was measured in the supernatant by reducing nitrate to nitrite with subsequent quantification of nitrite using the Griess reaction in which samples are compared to a standard curve of sodium nitrite was below the linear range of the assay.
iNOS and OvLV antigen were measured in MDM cells by immunofluorescent co-localization technique. Antibodies against OvLV capsid and iNOS were first tested for specific staining using chromagenic detection of OvLV infected MDM cytospin preparations (Fig. 6). Once the specific staining was confirmed, OvLV inoculated MDM cultures were examined over time for the percentage of cells producing virus antigen and evidence of co-localization with iNOS Table 1 Analysis of co-localization of OvLV and iNOS using a chi square test of a 2 2 contingency tablea
iNOS positive cell iNOS negative cells Total a
OvLV positive cells
OvLV negative cells
Total
400 (6%) 1514 (23%) 1914 (29%)
798 (12%) 3853 (59%) 4651 (71%)
1198 (18%) 5367 (82%) 6565 (100%)
The cell counts in each category were determined at 64 h PI when the cultures were treated with aminoguanidine. The row/ column association has a two-sided P value of 0.0004. The relative risk of OvLV positive and iNOS positive cells co-localizing is 1.184 (95% confidence interval ¼ 1:081–1.296).
(Fig. 6). The cumulative level of viral antigen positive cells exceeded background levels only in the latter portion of the experiment (64 and 72 h PI). Additionally, a rise above background in viral antigen associated fluorescence was only observed in the aminoguanidine treated cultures. Fluorescent co-localization was analyzed by the chi square test (Table 1). This test was only applied to time points PI in which the total viral antigen fluorescence was distinguished above autofluorescence levels. At 64 h PI, the relative risk of co-localization was calculated to be 1.184 (95% confidence interval ¼ 1:0811:296, P ¼ 0:0004).
4. Discussion This investigation provides two lines of evidence that nitric oxide negatively regulates lentivirus replication in MDMs: first, the addition of aminoguanidine to cultured macrophages infected with OvLV significantly increases the RT activity of the culture supernatant. Second, in a separate experiment, the addition of aminoguanidine to a similar culture system increased the production of OvLV capsid protein within
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Fig. 6. Relative co-localization of OvLV antigen (capsid) and iNOS in monocyte-derived macrophages (MDM). (A) L-Arginine treated and (B) immunofluorescent images of OvLV capsid (FITC-green) and iNOS (Cy3-red) in aminoguanidine treated MDM cell cultures at 72 h PI. All nuclei stained blue (DAPI). The white arrows point to multinucleated cells that are strongly positive for OvLV antigen expression. (C) Immunofluorescent co-localization of OvLV capsid antigen and iNOS in MDM cell cultures treated with aminoguanidine (AG) and L-arginine (LA). The bar graph represents the sum fluorescent area of OvLV capsid producing cells adjusted for cellular density (see Section 2 for formula). The black portions of the bars represent the area of fluorescence at each point that co-localized with iNOS production in the same cell. OvLV capsid expression was significantly ðP ¼ 0:01Þ above autofluorescence levels at 64 and 72 h PI only in the aminoguanidine treated culture.
the cytoplasm of infected macrophages. These two observations in the virus-infected cultures in which the production of nitric oxide is inhibited, suggest that the presence of nitric oxide may be an innate mechanism
of down regulating lentiviral replication following OvLVs initial replicative burst. RT activity was chosen as an indicator for viral replication as this enzyme is packaged within each
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virion during assembly. Thus, the quantity of RT activity in supernatant is presumed to be proportional to the number of virions in the supernatant. Since each well contained similar numbers of cells, the difference in RT activity was attributed to the treatments. The RT kit used was designed for quantitative analyses. Furthermore, RT activity was not detected sooner than 48 h after inoculation. Thus, the measured RT activity was from de novo virus production rather than carryover from the original inoculum. The interpretation that the presence of aminoguanidine increased RT activity is reinforced by a significant dose response curve. Finally, the addition of excess L-arginine, which competes directly with aminoguanidine for the active site of iNOS, partially counteracted the effects of the aminoguanidine. The experiments involving OvLV antigen production were performed to confirm that the level of RT activity was truly indicative of virus replication and refute the possibility that treatments were merely altering RT in vitro activity and not directly affecting virus replication. This alteration in RT activity could include direct interference, such as L-arginine inhibiting RT activity, or indirect interference with nitric oxide, reducing RT activity but not necessarily altering virus replication. The presence of virus antigen within MDM cell cytoplasm under the circumstances of this experiment is independent of RT activity. That is to say that although RT was necessary to initially create a provirus containing cell, that step was prior to any treatment and should be constant between aminoguanidine and L-arginine treated cultures. Conversely, the antigen measured in this experiment is viral capsid. The virus production was based on the number of cells containing virus capsid and not the activity of an enzyme. Thus, both the experiments involving measurement of RT activity and the experiment quantifying the number of OvLV antigen positive cells indicate that inhibition of nitric oxide synthase creates an environment which enhances OvLV replication. It was not entirely clear why the addition of exogenous nitric oxide did not further lower the RT activity of the treated cultures. One possible explanation is that a floor effect had been reached with the endogenous production of nitric oxide and addition of exogenous nitric oxide was not going to alter that lower RT activity. Alternatively, the half-life of the exogenously added nitric oxide (estimated by the
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manufacturer to be only a few minutes) might have been an insufficient exposure time to reduce viral replication as reflected in RT activity. As mentioned in Section 1, the regulation of iNOS in sheep has been difficult to define due to the low levels of nitrate accumulation in sheep cell cultures (Bogdan et al., 1997). This investigation also had difficulty in establishing functional relationships based on nitrate accumulation since the quantities of nitrite in the supernatants were outside the linear range of the Griess reaction to accurately measure changes. It is possible that measuring the levels of intracellular nitric oxide might resolve this investigative obstacle. The experimental design used herein does not address which step of viral replication may be altered by nitric oxide. Nitric oxide may influence viral replication at translation, transcription, virion packaging, or budding from the host cell. It is also possible that nitric oxide may act in concert with tumor necrosis factor-a (TNF-a) to influence viral replication since TNF-a is a potent mediator of nitric oxide. A further possibility exists that aminoguanidine may influence the nitric oxide to TNF-a ratio; thus indirectly altering viral replication (Rashid et al., 2001; Bacsi et al., 2001). Experiments addressing these questions would be a logical progression of this investigation. The results in the present investigation demonstrate the value of using sheep MDM cells to investigate mechanisms that affect lentivirus infection and replication. MDM cells were chosen over other cell populations such as alveolar macrophages because of their ease of acquisition and hardiness in culture. Although not directly measured in this investigation, it was presumed that the cell cultures were largely depleted of lymphocytes during the washing and media change steps. The MDM cells were strongly adhered to plastic culture surfaces whereas the lymphocytes are less so. Thus, intra-culture cytotoxicity from activated lymphocytes was presumed to not occur in this investigation. In conclusion, we believe that nitric oxide is part of the innate host response to lentivirus infection that down regulates OvLV replication in macrophages. The challenge that remains is to integrate our understanding of this innate response with the adaptive response of the host’s immune system to further define host– virus relationship.
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Acknowledgements This work was supported by NHLBI-NIH grant K08 HL 03792 to Dr. Kevin Keane.
References Akaike, T., 2001. Role of free radicals in viral pathogenesis and mutation. Rev. Med. Virol. 11, 87–101. Akaike, T., Maeda, H., 2000. Nitric oxide and virus infection. Immunology 101, 300–308. Bacsi, A., Csoma, E., Beck, Z., Andirko, I., Konya, J., Gergely, L., Toth, F.D., 2001. Induction of HIV-1 replication in latently infected syncytiotrophoblast cells by contact with placental macrophages: role of interleukin-6 and tumor necrosis factoralpha. J. Interferon Cytokine Res. 21, 1079–1088. Basu, A., Sehajpal, P.K., Ogiste, J.S., Lander, H.M., 1999. Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species. Antioxide Redox Signal 1, 105–112. Blacklaws, B.A., Bird, P., Allen, D., Roy, D.J., MacLennan, C.M., Hopkins, J., Sargan, D.R., McConnell, I., 1995. Initial lentivirus– host interactions within lymph nodes: a study of maedi-visna virus infection in sheep. J. Virol. 69 (3), 1400–1407. Blond, D., Cheret, A., Raoul, H., Le Grand, R., Caufour, P., Theodoro, F., Dormont, D., 1999. Nitric oxide synthesis during acute SIV mac251 infection of macaques. Res. Virol. 149, 75–86. Blond, D., Raoul, H., Le Grand, R., Dormont, D., 2000. Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages. J. Virol. 74, 8904–8912. Bogdan, C., 2001. Nitric oxide and the regulation of gene expression. Trends Cell Biol. 11, 66–75. Bogdan, J.R., Newlands-Monteith, C.F., Ellis, J.A., 1997. Nitric oxide production following in vitro stimulation of ovine pulmonary alveolar macrophages. Vet. Immunol. Immunopathol. 56, 299–310. Brodie, S.J., Marcom, K.A., Pearson, L.D., Anderson, B.C., de la Concha-Bermejillo, A., Ellis, J.A., DeMartini, J.C., 1992. Effects of virus load in the pathogenesis of lentivirus-induced lymphoid interstitial pneumonia. J. Infect. Dis. 166, 531–541. Brodie, S.J., Pearson, L.D., Zink, M.C., Bickle, H.M., Anderson, B.C., Marcom, K.A., DeMartini, J.C., 1995. Ovine lentivirus expression and disease virus replication, but not entry, is restricted to macrophages of specific tissues. Am. J. Pathol. 146, 250–263. Chebloune, Y., Karr, B., Sheffer, D., Leung, K., Narayan, O., 1996. Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep. J. Gen. Virol. 77, 2037–2051. Chen, F., Lu, Y., Castranova, V., Rojanasakul, Y., Miyahara, K., Shizuta, Y., Vallyathan, V., Shi, X., Demers, L.M., 1999. Nitric oxide inhibits HIV tat-induced NF-kappaB activation. Am. J. Pathol. 155, 275–284.
Chung, H.T., Pae, H.O., Choi, B.M., Billiar, T.R., Kim, Y.M., 2001. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun. 282, 1075–1079. Colasanti, M., Persichini, T., Venturini, G., Ascenzi, P., 1999. Snitrosylation of viral proteins: molecular bases for antiviral effect of nitric oxide. IUBMB Life 48, 25–31. DeMartini, J.C., Brodie, S.J., de la Concha-Bermejillo, A., Ellis, J.A., Lairmore, M.D., 1993. Pathogenesis of lymphoid interstitial pneumonia in natural and experimental ovine lentivirus infection. Clinical Infect. Dis. 17, 236–242. Duranton, C., Dorchies, P., Grand, S., Lesure, C., Oswald, I.P., 1999. Changing reactivity of caprine and ovine mononuclear phagocytes throughout part of the life cycle of Oestrus ovis: assessment through spontaneous and inductible NO production. Vet. Res. 30, 371–376. Duszyk, M., Radomski, M.W., 2000. The role of nitric oxide in the regulation of ion channels in airway epithelium: implications for diseases of the lung. Free Rad. Res. 33, 449–459. Gorrell, M.D., Brandon, M.R., Sheffer, D., Adams, R.J., Narayan, O., 1992. Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. J. Virol. 66, 2679–2688. Jeena, P.M., Coovadia, H.M., Thula, S.A., Blythe, D., Buckels, N.J., Chetty, R., 1998. Persistent and chronic lung disease in HIV-1 infected and uninfected African children. AIDS 12, 1185–1193. Jimenez, J.L., Gonzalez-Nicolas, J., Alvarez, S., Fresno, M., Munoz-Fernandez, M.A., 2001. Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide. J. Virol. 75, 4655–4663. Jungi, T.W., Thony, M., Brcic, M., Adler, B., Pauli, U., Peterhans, E., 1996. Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent. Immunobiology 195, 385–400. Jungi, T.W., Pfister, H., Sager, H., Fatzer, R., Vandevelde, M., Zurbriggen, A., 1997. Comparison of inducible nitric oxide synthase expression in the brains of listeria monocytogenesinfected cattle, sheep, and goats and in macrophages stimulated in vitro. Infect. Immun. 65, 5279–5288. Lairmore, M.D., Akita, G.Y., Russell, H.R., DeMartini, J.C., 1987. Replication and cytopathic effects of ovine lentivirus strains in alveolar macrophages correlate with in vivo pathogenicity. J. Virol. 61, 4038–4042. Lairmore, M.D., Poulson, J.M., Adducci, T.A., DeMartini, J.C., 1988. Lentivirus-induced lymphoproliferative disease. Comparative pathogenicity of distinct ovine lentivirus strains. Am. J. Pathol. 130, 80–89. Lander, H.M., 1996. Cellular activation mediated by nitric oxide. Methods 10, 15–20. Laskin, J.D., Laskin, D.L., 1999. Cellular and Molecular Biology of Nitric Oxide. Marcel Dekker, New York. Lechner, F., Schutte, A., Von Bodungen, U., Bertoni, G., Pfister, H., Jungi, T.W., Peterhans, E., 1999. Inducible nitric oxide synthase is expressed in joints of goats in the late stage of infection with caprine arthritis encephalitis virus. Clin. Exp. Immunol. 117, 70–75. Mankowski, J.L., Carter, D.L., Spelman, J.P., Nealen, M.L., Maughan, K.R., Kirstein, L.M., Didier, P.J., Adams, R.J.,
K.A. Keane et al. / Veterinary Immunology and Immunopathology 90 (2002) 179–189 Murphey-Corb, M., Zink, M.C., 1998. Pathogenesis of simian immunodeficiency virus pneumonia: an immunopathological response to virus. Am. J. Pathol. 153, 1123–1130. Mannick, J.B., Stamler, J.S., Teng, E., Simpson, N., Lawrence, J., Jordan, J., Finberg, R.W., 1999. Nitric oxide modulates HIV-1 replication. J. Acq. Immun. Def. Syn. 22, 1–9. Marcom, K.A., Pearson, L.D., Chung, C.S., Poulson, J.M., DeMartini, J.C., 1991. Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates. J. Clin. Microbiol. 29, 1472–1479. Mason, G.L., Yang, Z., Olchowy, T.W., Jian, Z., Bochsler, P.N., 1996. Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages. Vet. Immunol. Immunopathol. 53, 15–27. Mollace, V., Colasanti, M., Persichini, T., Bagetta, G., Lauro, G.M., Nistico, G., 1993. HIV gp120 glycoprotein stimulates the inducible isoform of no synthase in human cultured astrocytoma cells. Biochem. Biophys. Res. Commun. 194, 439–445. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D.E., Clements, J.E., 1983. Activation of caprine arthritis-encephalitis virus expression during maturation of monocytes to macrophages. Infect. Immun. 41, 67–73. Narayan, O., Zink, M.C., Gorrell, M., Crane, S., Huso, D., Jolly, P., Saltarelli, M., Adams, R.B., Clements, J.E., 1993. The lentiviruses of sheep and goats. In: Levy, J.A. (Ed.), The Retroviridae. Plenum Press, New York, pp. 229–255. Polazzi, E., Levi, G., Minghetti, L., 1999. Human immunodeficiency virus type 1 Tat protein stimulates inducible nitric oxide synthase expression and nitric oxide production in microglial cultures. J. Neuropathol. Exp. Neurol. 58, 825–831. Rashid, G., Luzon, A.A., Korzets, Z., Klein, O., Zeltzer, E., Bernheim, J., 2001. The effect of advanced glycation end-
189
products and aminoguanidine on TNFalpha production by rat peritoneal macrophages. Perit. Dial. Int. 21, 122–129. Sehajpal, P.K., Basu, A., Ogiste, J.S., Lander, H.M., 1999. Reversible S-nitrosation and inhibition of HIV-1 protease. Biochemistry 38, 13407–13413. Sherry, B., Schmidtmayerova, H., Zybarth, G., Dubrovsky, L., Raabe, T., Bukrinsky, M., 2000. Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response. Mol. Med. 6, 542–549. Sigurdsson, B., Grimsson, H., Pa´ lsson, P.A., 1952. Maedi, a chronic, progressive infection of sheep’s lungs. J. Infect. Dis. 90, 233–241. Southan, G.J., Szabo, C., 1996. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem. Pharmacol. 51, 383–394. Trifilieff, A., Fujitani, Y., Mentz, F., Dugas, B., Fuentes, M., Bertrand, C., 2000. Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression. J. Immunol. 165, 1526–1533. Wang, M.J., Jeng, K.C., Shih, P.C., 1999. Differential expression of inducible nitric oxide synthase gene by alveolar and peritoneal macrophages in lipopolysaccharide-hyporesponsive C3H/HeJ mice. Immunology 98, 497–503. Woodward, T.M., Carlson, J.O., de la Concha-Bermejillo, A., DeMartini, J.C., 1995. Biological and genetic changes in ovine lentivirus strains following passage in isogeneic twin lambs. J. Acq. Immun. Def. Syn. 8, 124–133. Zheng, J., Li, Y., Weiss, A.R., Bird, I.M., Magness, R.R., 2000. Expression of endothelial and inducible nitric oxide synthases and nitric oxide production in ovine placental and uterine tissues during late pregnancy. Placenta 21, 516–524.