Abstract
The effect of nitric oxide (NO) on Pneumocystis (Pc) organisms, the role of NO in the defense against infection with Pc, and the production of NO by alveolar macrophages (AMs) during Pneumocystis pneumonia (PCP) were investigated. The results indicate that NO was toxic to Pc organisms and inhibited their proliferation in culture. When the production of NO was inhibited by intraperitoneal injection of rats with the nitric oxide synthase inhibitor L-N5-(1-iminoethyl) ornithine, progression of Pc infection in immunocompetent rats was enhanced. Concentrations of NO in bronchoalveolar lavage fluids from immunosuppressed, Pc-infected rats and mice were greatly reduced, compared with those from uninfected animals, and AMs from these animals were defective in NO production. However, inducible nitric oxide synthase (iNOS) mRNA and protein concentrations were high in AMs from Pc-infected rats and mice. Immunoblot analysis showed that iNOS in AMs from Pc-infected rats existed primarily as a monomer, but the homo-dimerization of iNOS monomers was required for the production of NO. When iNOS dimerization cofactors, including calmodulin, were added to macrophage lysates, iNOS dimerization increased, whereas incubation of the same lysates with all cofactors except calmodulin did not rescue iNOS dimer formation. These data suggest that NO is important in the defense against Pc infection, but that the production of NO in AMs during PCP is defective because of the reduced dimerization of iNOS.
Keywords: Pneumocystis, nitric oxide, iNOS, alveolar macrophage, calmodulin
CLINICAL RELEVANCE.
This research reveals the mechanism by which infection with Pneumocystis causes a defect in the production of nitric oxide in alveolar macrophages. This information will enable the development of methods to prevent this defect and enhance host defense ability against infection with Pneumocystis.
Complex interactions between host and pathogen occur during Pneumocystis pneumonia (PCP), triggering both innate and adaptive immune responses. Studies showed that the alveolar macrophage (AM) is an important cell for the clearance of Pneumocystis organisms from the lungs during PCP (1, 2). The clearance of organisms by AMs is mediated by direct phagocytosis (2–4), the production of toxic metabolites (5, 6), and the modulation of inflammatory cytokines (2–4). Some of these functional abilities are defective (7, 8), and the number of AMs (7, 9) is decreased during PCP. The reasons for the reduced function of AMs are largely unknown.
AMs are an important source of nitric oxide (NO) in the lung during infection (10). NO is a short-lived, freely diffusible molecule with many functions in normal physiology (11) and in response to cytokines (12), infectious agents, or bacterial products (13). It has an antimicrobial effect on organisms such as Toxoplasma gondii (14), Leishmania donovani (15), and Cryptococcus neoformans (16). NO also causes a redistribution of the actin pool in some cells (17). This reorganization is mediated by calmodulin, because the inhibition of calmodulin activity suppresses redistribution, despite high concentrations of NO (17). Calmodulin was shown to be an important molecule for the survival of AMs, but its concentration in AMs is decreased during PCP (18).
Pneumocystis (Pc) organisms were shown to stimulate AMs from humans and rats to produce NO (19), and both chemically produced NO (20) and IFN-γ–stimulated NO (21) are toxic to Pc organisms. However, elevated concentrations of inducible nitric oxide synthase (iNOS) mRNA and protein in AMs in Pc-infected, CD4+ cell–depleted mice did not reduce the organism burden or slow disease progression (22, 23). In this study, we investigated this problem further, and found that the inability of the iNOS protein to form dimers was a cause of this defect.
MATERIALS AND METHODS
Reagents
(Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate (Spm NONOate), diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate), and L-N5-(1-iminoethyl)ornithine (L-NIO) were purchased from Cayman Chemical (Ann Arbor, MI). Tetrahydrobiopterin (BH4) was purchased from Alexis Biochemicals (Lausen, Switzerland). Heme was obtained from MP Biochemicals (Irvine, CA), and L-arginine hydrochloride was purchased from Fisher Bioreagents (Pittsburgh, PA). Anti-rat and anti-mouse iNOS antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was obtained from US Biological (Swampscott, MA). Calmodulin protein was purchased from ANIARA (Manson, OH). All other reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), unless stated otherwise.
Short-Term Culture of Pc Organisms
Pc organisms were grown on monolayers of human embryonic lung (HEL 299) cells (CCL-137; ATCC, Manassas, VA) on 24-well culture dishes in 0.5 ml minimum essential medium containing 2 mM L-glutamine, 17.9 mM sodium bicarbonate, 0.1 mM nonessential amino acids, 10% FCS, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C and 5% O2, 10% CO2, and 85% nitrogen. Pneumocystis carinii inoculum was isolated and added to cultures as previously described (24).
Rodent Models of PCP
The immunosuppression of rats with dexamethasone (7) and of mice with anti-CD4 antibody (1, 25), the transtracheal inoculation of animals with Pc organisms (7), bronchoalveolar lavage (BAL) (26), and the isolation of AMs from BAL fluids (7) were performed as previously described.
Infection of Mice with Histoplasma capsulatum
Histoplasma capsulatum (IU-CT) yeasts were grown in Histoplasma Macrophage Medium (HMM) broth, as described previously (27). Six-week-old C57/BL6 mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized with 5% isoflurane and intranasally instilled with 50 μl inoculum containing 1 × 106 yeast of H. capsulatum. At 7 and 14 days after infection, BAL fluids and AMs were obtained from these mice by BAL.
Assessment of Nitrate/Nitrite Concentrations
The nitrite assay was performed using a NO Quantitation Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.
RNA Isolation and Real-Time RT-PCR
Total RNA was isolated from AMs using the TRIzol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Real-time RT-PCR for rat or murine iNOS was performed using Assays-on-Demand Gene Expression Kits (Applied Biosystems, Foster City, CA) on a Smartcycler (Cepheid, Sunnyvale, CA). Ribosomal protein S8 (RPS8) mRNA was assayed in an identical manner as a control, as described elsewhere (28). Organism viability was estimated based on the ribosomal RNA levels of Pc mitochondrial large-subunit ribosomal RNA gene, as described elsewhere (29).
Immunoblot Blot Analysis of iNOS Protein
Determinations of protein concentration and Western blotting were performed as described elsewhere (30). For the determination of iNOS dimer formation, electrophoresis was performed under partially denatured conditions to allow the iNOS dimer to remain intact, as previously described (31). Immunoblot autoradiographs were analyzed using the ImageJ software package, which is a Java-based image processing program developed at the National Institutes of Health (32).
Statistical Analysis
Comparisons between the mean values of treatment and control groups and data regarding the effects of NO on Pc proliferation over time were analyzed using the Mann-Whitney U test. P < 0.05 was considered significant.
RESULTS
Production of NO by Chemical Generators in Culture Media, and Effects of NO on Pc Viability
Spm NONOate and DEA NONOate were incubated in complete medium, and the release of NO from these chemicals was induced by decreasing the pH of the medium from 8.0 to 7.4 with 1 N HCl. At pH 7.4 and 37°C, the half-life of DEA NONOate is 2.1 minutes, and that of Spm NONOate is 39 minutes (product information; Cayman Chemical). The pH of medium in a well of each condition was checked daily and maintained at 7.0–7.4. Both chemicals produced NO in a dose-dependent manner. The production of NO (measured as nitrites) by DEA NONOate (0.1 mM) peaked (20.2 ± 2.0 μM) at 15 minutes after lowering the pH; little NO was produced after that time. The production of NO by Spm NONOate (0.1 mM) was still increasing 60 minutes after the change in pH (23.7 ± 1.8 μM at 60 minutes). Lower concentrations (0.01 mM) of Spm NONOate and DEA NONOate generated roughly 30% as much NO as the higher concentrations (6.9 ± 1.2 μM for Spm NONOate, and 6.5 ± 2.0 μM for DEA NONOate). The times for peak production were the same as those for the higher concentrations of each compound. All values shown are means ± SDs.
To test the toxicity of these compounds, a half million P. carinii organisms were incubated with carrier (0.01 mM NaOH) or NO generators, and then assessed for viability by measuring Pc mitochondrial large-subunit ribosomal RNA concentrations at 2 or 4 hours (Figure 1). Compared with carrier-treated control concentrations at 4 hours, 0.01 mM Spm NONOate decreased Pc viability by 17%, whereas 0.1 mM Spm NONOate reduced it by 33% at 2 hours and 43% at 4 hours (Figure 1). Pc organisms incubated with 0.01 mM or 0.1 mM DEA NONOate did not show significant decreases in viability at either time point, probably because of the shorter duration of NO production by DEA NONOate.
Figure 1.
Effect of NO on Pneumocystis organisms. P. carinii organisms were incubated with 0.01 or 0.1 mM (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate (Spm NONOate) or diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) for 2 or 4 hours, and then assayed for viability by real-time RT-PCR for P. carinii mitochondrial large-subunit ribosomal RNA. *P < 0.05 versus carrier (0.01 mM NaOH) controls at the same time point. $P < 0.05 versus same condition at 2 hours.
Suppression of Pc Proliferation in Short-Term Culture by NO
Pc was cultured on HEL cell monolayers. High (0.1 mM) and low (0.01 mM) concentrations of NO generators were included in the culture media and replenished every 24 hours. Separate cultures treated with carrier (0.1% DMSO in saline) or trimethoprim/sulfamethoxazole (Tmp/Smx, 50/250 μg/ml) were used as negative and positive controls, respectively. The cultures were examined under an inverted microscope every other day to assess the health of the monolayer. Any culture showing signs of contamination by other microorganisms (bacteria or fungi) was discarded. Organisms were sampled and counted every other day, as described previously (24). Untreated cultures showed a significant increase in numbers of organisms after 7 days of culture (21.3 ± 4.1 organisms per ×1,000 microscopic field; Figure 2). Tmp/Smx inhibited the propagation of organisms in culture by 89% (2.4 ± 0.4 organisms per ×1,000 microscopic field; P < 0.0001 versus untreated controls) at the same time point (7 days). DEA NONOate at either concentration and Spm NONOate at 0.01 mM exerted minimal effects (Figure 2), whereas Spm NONOate at 0.1 mM suppressed 97% of Pc growth at 7 days (0.58 ± 0.1 organisms per ×1,000 microscopic field). These results indicate that the NO generated was inhibitory to Pc replication in short-term culture.
Figure 2.
Inhibitory effect of NO on proliferation of Pneumocystis (Pc) in short-term culture. P. carinii organisms were inoculated onto human embryonic lung cell monolayers in 24-well plates, and numbers of organisms in cultures were counted every other day. Some wells contained trimethoprim/sulfamethoxazole (Tmp/Smx; 50/250 μg/ml) or the nitric oxide generators Spm NONOate or DEA NONOate at 0.1 mM or 0.01 mM, and the media were adjusted to pH 7.4 daily. Results are averages ± SDs of quadruplicate wells of each condition in five independent experiments.
To control for the toxicity of NO to HEL cells, the health of the monolayer was monitored in each condition. Without Pc, HEL cell culture alone exhibited only 5.4% ± 1.0% cell death, but 54.6% ± 6.6% of Pc-infected cells died (P < 0.05 versus controls) after 7 days of culture. Spm NONOate at 0.1 mM inhibited 97% of Pc growth, as already mentioned, but caused only 10.7% ± 2.3% cell death by Day 7, indicating that the suppression of Pc growth in cultures by NO generators was not attributable to damage of the cell monolayer by NO.
Increased Pc Proliferation In Vivo by Suppressing the Production of NO
To determine if NO participates in the control of Pc proliferation in vivo, uninfected (Normal) and Pc-inoculated Normal (Normal-Pc) rats were injected (intraperitoneally) daily with L-NIO at 2 mg/kg to inhibit the production of NO, and then assessed for organism burden and NO concentrations in AMs and BAL fluid. NO is quickly converted to nitrites and nitrates in vivo (33–35). Nitrates can be converted to nitrites through the action of nitrate reductase. Therefore, NO concentrations in AMs and BAL fluid were determined by measuring nitrites after the conversion of nitrates to nitrites, using nitrate reductase in a NO quantitation kit. AMs were isolated, incubated for 1 hour in saline, lysed by sonication, and assayed for nitrite concentrations.
AMs from rats treated with L-NIO for 1 week produced 1.5 ± 0.2 μM (normalized to 1 mg of AM protein) of nitrites. This amount was 62% less than that (3.9 ± 0.5 μM) produced by AMs from untreated rats (P < 0.05; Figure 3). The ability to produce NO by AMs was further impaired with longer treatment of L-NIO. After 2 weeks of L-NIO injections, the production of nitrites in AMs decreased by 73% (1.0 ± 0.1 μM versus 3.7 ± 0.2 μM for untreated controls; P < 0.05; Figure 3).
Figure 3.
Effect of L-N5-(1-iminoethyl)ornithine (L-NIO) on alveolar macrophage (AM) nitrite concentrations and Pneumocystis burden. (A) Uninfected (Normal) and Pc-challenged (Normal-Pc) immunocompetent rats received intraperitoneal injections daily with 2 mg/kg of L-NIO, starting 2 days after inoculation with Pc. AMs were isolated from these rats 1 or 2 weeks after initiation of L-NIO treatment, and assessed for nitrite concentrations with or without stimulation of the AMs with 10 ng/ml IFN-γ and 5 × 106 Escherichia coli. Nitrite concentrations are expressed in μM relative to 1 mg AM protein in cell lysate. (B) Numbers of Pneumocystis organisms (both trophic and cyst forms) in Normal-Pc rats after L-NIO treatment were determined by examining Giemsa-stained lung impression smears. Numbers of organisms consist of average counts from 10 microscopic fields at ×1,000 magnification. *P < 0.05, compared with untreated group.
The response of AMs to stimulation by IFN-γ and Escherichia coli organisms to produce NO was then assessed. One hour of incubation with 10 ng/ml IFN-γ and 5 × 106 E. coli organisms increased the production of nitrites in AMs from Normal rats by approximately threefold (from 3.9 ± 0.5 μM to 11.6 ± 2.2 μM for those treated with carrier for 1 week, and from 3.7 ± 0.2 to 10.2 ± 1.1 μM for those treated with carrier for 2 weeks). In contrast, AMs from L-NIO–treated rats increased the production of nitrites by only 1.4-fold and 1.2-fold, respectively, from 1.5 ± 0.2 μM to 2.1 ± 0.4 μM for those treated for 1 week, and from 1.0 ± 0.1 μM to 1.2 ± 0.2 μM for those treated for 2 weeks (Figure 3). AMs from Normal-Pc rats that had been inoculated with Pc for 1 week or 2 weeks produced significantly more nitrites (14.7 ± 2.0 μM and 15.3 ± 1.8 μM, respectively; P < 0.05, versus controls at the same time point). Stimulation with IFN-γ and E. coli did not increase the concentrations of nitrites produced by AMs at either time point.
In contrast to the increase in nitrite production in Normal rats inoculated with Pc, AMs from L-NIO–treated Normal-Pc rats did not show an increase in the production of nitrites in response to Pc inoculation (3.2 ± 0.4 μM after 1 week, and 2.3 ± 0.2 μM after 2 weeks of L-NIO treatment; P < 0.05 for each, versus Normal-Pc rats). IFN-γ and E. coli also failed to stimulate further production of nitrites in these cells (3.0 ± 0.3 μM for 1 week, and 2.1 ± 0.3 μM for 2 weeks; Figure 3).
Organism burdens in Normal-Pc rats with or without L-NIO treatment were also determined. After 1 week of infection, impression smears of lungs from Normal-Pc rats showed an average of 0.6 ± 0.1 trophic forms and 0.1 ± 0.1 cysts per ×1,000 microscopic field (Figure 3). Rats treated with L-NIO showed higher burdens, with an average of 1.9 ± 0.2 trophic forms and 0.2 ± 0.1 cysts at the same time point. These represent 3.1-fold and twofold increases in trophic forms and cysts, respectively, and both increases were statistically significant (P < 0.05 versus Normal-Pc). At 2 weeks, the difference in organism burden between untreated and L-NIO–treated rats was even larger. Normal-Pc rats had begun to clear the organisms, and both trophic form (0.5 ± 0.1) and cyst (0.1 ± 0.1) numbers were reduced or unchanged. In contrast, organism burdens in L-NIO–treated rats had increased from 1.9 ± 0.2 trophic forms/field to 3.6 ± 0.2 trophic forms/field, and from 0.2 ± 0.1 cysts/field to 0.6 ± 0.1 cysts/field at 1 week (P < 0.0001 versus Normal-Pc and Normal-Pc + L-NIO; Figure 3). These results demonstrate that AMs do produce NO in response to infection with Pc, and that the inhibition of NO production hampers the clearance of Pc from the lungs of immunocompetent rats. Because L-NIO–treated rats were not immunosuppressed, they all cleared the infection and survived.
Low Nitrite Concentrations in BAL Fluids of Pc-Infected Rats and Mice
To investigate the production of NO in the lung during PCP, the first 5 ml of BAL fluids from uninfected and Pc-infected immunosuppressed rats were assayed for nitrite concentrations. The results showed that BAL fluids from dexamethasone (Dex)-Pc rats contained 35% less nitrites than those from either Normal or Dex rats (P < 0.05; Table 1). BAL fluids from Normal-Pc rats had threefold more nitrites than those from Normal rats that were not inoculated with Pc, and 4.5-fold more than those from Dex-Pc rats (P < 0.05; Table 1).
TABLE 1.
NITRITE CONCENTRATIONS IN BALF AND AMs FROM RATS AND MICE WITH PCP*
| Sample | Animal | Normal | Suppressed† | 4 Weeks of PCP | 7 Weeks of PCP | IFN-γ/Eschrichia coli‡ | Normal-Pc§ |
|---|---|---|---|---|---|---|---|
| BALF | Rat | 3.2 ± 0.2 | 3.4 ± 0.6 | NA | 2.1 ± 0.3* | NA | 9.6 ± 1.3# |
| Mouse | 2.3 ± 0.4 | 2.2 ± 0.2 | 1.3 ± 0.1¶ | 1.0 ± 0.1* | NA | NA | |
| AMs | Rat | 4.2 ± 0.3 | 4.5 ± 0.4 | NA | 0.8 ± 0.1* | 1.0 ± 0.1¶ | 15.6 ± 1.2# |
| Mouse | 4.8 ± 0.4 | 4.3 ± 0.3 | 2.0 ± 0.2¶ | 1.7 ± 0.2* | 2.1 ± 0.3¶ | NA |
Definition of abbreviations: AMs, alveolar macrophages; BALF, bronchoalveolar lavage fluid; NA, not assessed; Pc, Pneumocystis; PCP, Pneumocystis pneumonia. All values are mean ±SD.
BALF and AM lysates were treated with nitrate reductase to convert nitrates to nitrites before the determination of nitrite concentrations. Nitrite concentrations in the first 5 ml rat BALF or the first 1 ml mouse BALF are expressed in μM, and those in AMs (1 × 106 from rats, and 5 × 105 from mice) are expressed in μM relative to 1 mg of protein in cell lysates.
Rats were immunosuppressed with dexamethasone, and mice were immunosuppressed with anti-CD4 antibody.
Preincubated with 10 ng/ml IFN-γ and 5 × 106 Escherichia coli for 1 hour before assay.
Normal rats challenged with P. carinii.
P < 0.05 versus immunosuppressed control samples.
P < 0.05 versus normal control samples.
Previous results indicate that CD4-depleted–Pc mice respond differently from rats in the timing of decreases in numbers of AMs (9). Therefore, BAL fluid (first 1 ml) nitrites were assessed at both 4 and 7 weeks of infection, and were found to contain 41% (1.3 ± 0.1 μM) and 55% (1.0 ± 0.2 μM), respectively, less nitrites than those from CD4-depleted mice (P < 0.05; Table 1). These data suggest that the decrease in nitrites in BAL fluid during PCP is not species-specific, and is not related to the method of immunosuppression.
Decreased Nitrite Concentrations in AMs during PCP in Rats and Mice
To assess the production of NO by AMs from rats and mice with PCP, AM-produced nitrites were measured after 1 hour of culture. Nitrite concentrations were first assayed in AM samples treated with saline alone, to determine background values of nitrites for the assay. AMs from Dex-Pc rats (n = 15) were found to produce only 20% as much NO (P < 0.05 versus Dex controls; Table 1) as those from Normal or Dex rats (n = 12 each). Stimulation of these cells with IFN-γ and E. coli did not significantly increase nitrite concentrations. Likewise, CD4-depleted–Pc mice from early (4-week) and late (7-week) infections contained significantly lower nitrite concentrations (P < 0.05; Table 1; n = 15–20). These results indicate that AMs from PCP rats and mice are defective in the production of NO.
Production of NO in AMs from H. capsulatum–Infected Mice
To determine whether the defect in NO production by AMs is specific to infection with Pc, mice were infected with the yeast form of H. capsulatum by intranasal instillation. At 7 days (mid-stage) and 14 days (late stage) after infection, nitrite concentrations in AMs and BAL fluid were measured. The nitrite concentrations in both AMs and BAL fluid from mice infected for 7 days exhibited no significant difference, compared with uninfected controls. However, 5.56-fold and 4.62-fold increases in nitrite concentrations in AMs and BAL fluid, respectively, were evident 14 days after infection (Table 2). These results suggest that in contrast to infection with Pc, the production of NO in AMs increases during H. capsulatum infection.
TABLE 2.
NITRITE CONCENTRATIONS IN BALF AND AMs FROM H. CAPSULATUM-INFECTED MICE
|
H. capsulatum-Infected |
|||
|---|---|---|---|
| Uninfected | Day 7 | Day 14 | |
| BALF | 0.29 ± 0.35 | 0.26 ± 0.41 | 1.34 ± 0.34* |
| AMs | 0.18 ± 0.20 | 0.10 ± 0.32 | 1.00 ± 0.12* |
BALF and AM lysates were treated with nitrate reductase to convert nitrates to nitrites before the determination of nitrite concentrations. Nitrite concentrations in the first 1 ml BALF are expressed in μM, and those in AMs (5 × 105) are expressed in μM relative to 1 mg of protein in the cell lysates. All values are mean ± SD.
P < 0.05 versus uninfected control.
iNOS Transcription in PCP Rats and Mice
Real-time RT-PCR for iNOS mRNA concentrations was performed to determine if the defect in NO production was attributable to the reduced transcription of the iNOS gene. RNAs from two million AMs from Normal, Dex, and Dex-Pc rats, or one million AMs from Normal, CD4-depleted, or CD4-depleted–Pc mice were converted to cDNAs and used for the real-time RT-PCR. As shown in Figure 4, iNOS transcripts, normalized to RPS8, were 14.6- fold higher in AMs from Dex-Pc rats than in those from Dex rats (P < 0.05). This finding is in direct contrast to the low nitrite concentrations in AMs from Dex-Pc rats (Table 1). iNOS mRNA concentrations in AMs from Normal and Dex rats were very similar, indicating that immunosuppression by dexamethasone did not significantly affect iNOS transcription in AMs. AMs from Normal-Pc rats had increased iNOS transcripts, compared with those from Normal rats (14.7-fold; P < 0.05). As with rats, real-time RT-PCR results indicated that AMs from CD4-depleted–Pc mice had higher concentrations (13.6-fold) of iNOS mRNA than those from CD4-depleted mice (Figure 4). These results indicate that the upregulation of iNOS transcription in AMs is a normal immune response to Pc.
Figure 4.
Inducible nitric oxide synthase (iNOS) mRNA concentrations in AMs during Pneumocystis pneumonia (PCP). Concentrations of iNOS mRNA relative to those of control gene ribosomal protein S8 (RPS8) in AMs from Pneumocystis-infected rats (A) and mice (B) were determined by real-time RT-PCR. Dexamethasone (Dex)-Pc AMs were from rats that had been infected with P. carinii for 6 weeks. CD4-depleted–Pc (CD4-depl–Pc) AMs were from mice infected with P. murinii for 4 weeks. Normal-Pc AMs were isolated from normal rats 17 days after inoculation with P. carinii. Results represent mean fold change ± SD relative to normal controls from 4–7 animals in each group. *P < 0.05 versus immunosuppressed group (Dex or CD4-depl). #P < 0.05 versus Normal group.
iNOS Protein Concentrations in Rats and Mice with PCP
To determine whether the iNOS mRNA detected in AMs from rats was translated, Western immunoblotting was performed. Equal amounts of AM-soluble protein from Normal, Dex, and Dex-Pc rats were electrophoresed on a polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was reacted with antibody against the iNOS protein, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. The iNOS protein was detected in all samples (Figure 5A), and AMs from Dex-Pc rats were found to produce significantly more iNOS protein than AMs from Normal or Dex rats.
Figure 5.
iNOS protein concentrations in AMs from PCP rats and mice. (A) Total soluble protein from AMs of Normal, Dex, and Dex-Pc rats was separated by SDS-PAGE and reacted with an anti-iNOS antibody. (B) AM proteins from Normal, CD4+ lymphocyte–depleted (CD4-depl), and CD4-depleted Pc–infected (CD4-depl-Pc) mice were treated as described for rat AMs in A. CD4-depl-Pc mice were infected with P. murina for 4 or 7 weeks before being killed. The iNOS protein shown here is the 130-kD monomer. Results are representative of four independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
The CD4-depleted murine model of PCP was also assessed for the expression of iNOS protein. As shown in Figure 5B, the amount of iNOS protein was low in AMs from Normal and CD4-depleted mice, but AMs from those infected with P. murina for either 4 or 7 weeks contained significantly increased concentrations of iNOS protein.
iNOS Dimerization in AMs during PCP
To determine if the decreased production of NO by AMs was attributable to reduced iNOS dimerization, partially denaturing immunoblotting was performed as described previously (31) to identify both the iNOS dimmer (260 kD) and monomer (130 kD) in AM lysates. As shown in Figure 6A, pooled AMs from three Normal or Dex rats had approximately equal levels of iNOS monomer and dimer. This ratio was shifted in AMs from Dex-Pc rats with high concentrations of the iNOS monomer and low concentrations of the iNOS dimer (Figure 6A). Image analysis of the immunoblots showed that Normal and Dex animals dimerized 46–58% of their AM iNOS (Figure 6B). In contrast, AMs from Dex-Pc rats dimerized only 15% of their iNOS protein (Figure 6B).
Figure 6.
iNOS dimer formation in rat AMs during PCP. (A) Total soluble protein from rat AMs was separated by SDS-PAGE under partially denaturing conditions, as previously described (31), and subjected to Western blotting with an anti-iNOS antibody. Pooled AMs from three Normal and Dex rats and five Dex-Pc rats were used. The molecular weights of the iNOS monomer and dimer are 130 and 260 kD, respectively. Image analysis of four independent Western blots was performed by normalizing iNOS monomer and dimer signals to that of the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Average iNOS dimer concentrations ± SDs are plotted as a percentage of iNOS monomer concentrations. *P < 0.05, versus Dex control samples.
Effects of Cofactors on iNOS Dimerization
To determine whether the low iNOS dimer concentrations were attributable to a reduced availability of cofactors necessary for iNOS dimerization, AM lysates from Normal, Dex, or Dex-Pc rats were incubated for 30 minutes with iNOS dimerization cofactors in sterile saline containing 1 mM PMSF before being analyzed for iNOS dimers. These cofactors included heme, tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide phosphate (NADPH), arginine, and in some conditions, calmodulin. The concentrations of cofactors used were similar to those previously reported (36, 37).
Normal or Dex AM lysates, supplemented with all cofactors, showed similar concentrations of iNOS dimers (Figure 7A). For Dex-Pc AM lysates, concentrations of iNOS monomers were high and those of iNOS dimers were low without the presence of cofactors. The addition of all cofactors significantly increased the concentrations of iNOS dimers, whereas the concentrations of monomers decreased (Figure 7A). However, when calmodulin was not included among the cofactors, Dex-Pc AM lysates contained very low concentrations of iNOS dimers (Figure 7A). Image analysis of three independent immunoblots (Figure 7B) showed that 17.9% ± 1.9% of iNOS proteins were dimerized without the addition of any cofactors, and that 18.4% ± 2.1% of iNOS proteins were dimerized when the cell lysate was incubated with all cofactors except calmodulin. In contrast, 62.0% ± 9.8% of iNOS proteins were dimerized when the cofactors included 5 μM calmodulin (Figure 7B).
Figure 7.
iNOS dimer formation in AM lysates after incubation with cofactors and calmodulin. (A) AMs from Normal (n = 3), Dex (n = 3), and Dex-Pc (n = 5) rats were pooled and sonicated on ice. AM lysates were incubated with iNOS dimerization cofactors, including heme (10 μM), tetrahydrobiopterin (10 μM), flavin adenine dinucleotide (FAD) (4 μM), flavin mononucleotide (FMN) (4 μM), nicotinamide adenine dinucleotide phosphate (NADPH) (2 mM), and arginine (2 mM), with or without calmodulin (5 μM), in sterile saline solution containing 1 mM PMSF. After 30 minutes of incubation, cell lysates were subjected to partially denaturing Western blot analysis, as described in the legend for Figure 6. (B) Histogram represents average iNOS dimerization levels ± SDs from three independent trials. *P < 0.05 versus Dex controls.
DISCUSSION
To survive in the lung, Pc must evade the actions of AMs. We previously showed that infection with Pc renders AMs defective in phagocytosis (8) and induces AM apoptosis (1, 7, 9). In this study, we found that AM defense is further weakened during infection with Pc because of decreased NO production. We first demonstrated that Pc is susceptible to NO produced in vitro by the NO generators, Spm NONOate or DEA NONOate. Spm NONOate at a concentration of 0.1 mM also inhibited the proliferation of Pc in culture on HEL monolayers (Figure 2). This inhibition was attributable to effects of NO on the organism rather than on the feeder HEL cells. These data agree with previous results on the toxicity of NO to Pc (20, 21), and confirm that its action is directed against the organism.
The production of NO by AMs is mediated by iNOS, and the regulation of iNOS expression occurs primarily at the transcriptional level. Therefore, Shellito and colleagues determined iNOS mRNA and protein concentrations in AMs from uninfected and Pc-infected CD4 cell–depleted mice and severe combined immunodeficiency (SCID) mice, to investigate the role of NO in host defense against infection with Pc (23). Because the organism burden remained high despite elevated concentrations of iNOS mRNA and protein in the AMs of these mice, they concluded that iNOS is an unlikely participant in host defense against Pc, and speculated that NO is not important in this regard, but they did not measure concentrations of NO or nitrites (23). In the present study, we also found that concentrations of both iNOS mRNA and protein had increased in AMs from Pc-infected animals (Figures 4 and 5), but the production of NO by these cells was defective (Table 1). This finding explains why the organism burden was high even though iNOS concentrations were high. The iNOS detected by Shellito et al. (23) may not have been functional. Therefore, insufficient NO was produced to kill the organisms. The defect in NO production was not evident in AMs from H. capsulatum–infected mice (Table 2). Because H. capsulatum can infect immunocompetent animals, these H. capsulatum–infected mice were not immunosuppressed. Therefore, whether host immunosuppression or Pc infection itself is responsible for the decreased production of NO in AMs remains to be confirmed.
We found that AMs from immunocompetent rats challenged with Pc responded to the organism by producing large amounts of NO, and the concentrations remained high for 2 weeks while the animals were clearing the organisms. These data suggest that the production of NO is a normal host response to infection with Pc in immunocompetent animals. This possibility is supported by the observation that lower concentrations of NO in L-NIO–treated Normal-Pc rats were accompanied by increased organism burdens (Figure 3). The increase in organism burden was more pronounced after 2 weeks of treatment with L-NIO, suggesting that NO plays a role in the control of Pc proliferation during the normal immune response.
When AMs from normal rats were stimulated with IFN-γ and E. coli, the production of NO increased approximately threefold (Table 1). Surprisingly, no additional production of NO was evident when AMs from normal rats challenged with Pc were treated with IFN-γ and E. coli. This lack of response to stimulation with IFN-γ and E. coli in the production of NO was also evident in AMs from Pc-infected rats (Table 1). These results suggest that infection with Pc can render the production of NO in AMs defective. This is confirmed by the findings that AMs from Pc-infected rats produced very low concentrations of NO, and that these AMs were also unresponsive to stimulation with IFN-γ and E. coli (Table 1). The expression of iNOS in AMs may be maximally induced during infection with Pc. Therefore, additional response does not occur when the cells are stimulated with IFN-γ or E. coli.
The decrease in production of NO in Dex-Pc rats was not attributable to dexamethasone, because the antibody-mediated immunosuppression of mice by the depletion of CD4+ cells revealed the same phenomenon. Previous studies indicate that these mice undergo a transient increase in AM number before a profound decrease by 7 weeks of infection (9). The low nitrite concentrations in murine lungs at 4 weeks of infection, when the number of AMs is maximal, suggest that the production of NO in AMs is suppressed even at early stages of infection.
In addition to the regulation of transcription, the production of iNOS can also be regulated at the translational or posttranslational level. Transforming growth factor–β can promote the degradation of iNOS protein and decrease translation of the transcript (36). Imidazole-based antifungal agents (37) and carbon monoxide (38) can inhibit the dimerization of iNOS or the activity of the dimer. Cofactor availability and binding also modulate the stability of iNOS, because the suppression of BH4 by cytokines results in reduced iNOS dimerization (39). In this study, iNOS protein synthesis increased during PCP (Figure 5), indicating that the reduction in NO production during PCP was not attributable to defects in the production of the iNOS protein.
iNOS must homo-dimerize to form a functional enzyme and requires the cofactors BH4, heme, arginine, NADPH, FAD+, FMN, and calmodulin for this action (40, 41). iNOS converts arginine to citrulline and NO (40, 42). If arginine concentrations are low, the iNOS protein will not dimerize (41). The dimerization of iNOS is initiated by the binding of calmodulin, FAD+, and FMN to the monomer, followed by the addition of a heme molecule near the N-terminus of the monomer. This stimulates the addition of another iNOS monomer, which is stabilized by arginine and BH4 (41). The results of our study showed that the dimerization of iNOS monomers occurs normally in AMs from uninfected and Pc-challenged immunocompetent animals, but is reduced in AMs from Dex-Pc rats (Figures 6 and 7).
The observation that the addition of calmodulin and other cofactors, but not the other cofactors alone, rescued iNOS dimer formation (Figure 7) suggested that the available calmodulin is insufficient in AMs during PCP. This conclusion is supported by our recent results indicating that calmodulin mRNA and protein concentrations are significantly decreased in AMs during PCP (18). Calmodulin is a ubiquitous calcium-sensing protein. The binding of calcium to calmodulin results in an altered conformation and permits it to bind other proteins. iNOS dimerization requires apocalmodulin (calcium-free calmodulin) as a cofactor (40, 43).
The results of this study suggest that the production of NO is an important host response to Pc, and that the defect in iNOS dimerization confers a survival advantage to the organism. AMs attempt to increase the production of NO by upregulating the expression of iNOS, but fail to achieve this goal because of the decreased availability of calmodulin for dimerization. Because calmodulin downregulation also contributes to AM defects in survival and granulocyte macrophage colony-stimulating factor (GM–CSF) signaling (18), additional research is required to define the mechanisms that prevent calmodulin downregulation and restore iNOS dimerization. Although the inhalation of NO in conjunction with Tmp/Smx and prednisolone was used to treat PCP (44, 45), the roles of iNOS and NO in the defense against Pc infections in humans are not known. This animal study will serve as a model for further studies on the involvement of NO in human PCP.
Originally Published in Press as DOI: 10.1165/rcmb.2009-0367OC on June 17, 2010
The study was supported by National Institutes of Health grants RO1 HL65170 and R01 AI062259 (C.-H.L.).
Author Disclosure: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
References
- 1.Lasbury ME, Durant PJ, Ray CA, Tschang D, Schwendener R, Lee CH. Suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with Pneumocystis pneumonia. J Immunol 2006;176:6443–6453. [DOI] [PubMed] [Google Scholar]
- 2.Limper AH, Hoyte JS, Standing JE. The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung. J Clin Invest 1997;99:2110–2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Ezekowitz RAB, Williams DJ, Koziel H, Armstrong MY, Warner A, Richards FF, Rose RM. Uptake of Pneumocystis carinii mediated by the macrophage mannose receptor. Nature 1991;351:155–158. [DOI] [PubMed] [Google Scholar]
- 4.Masur H, Jones TC. The interaction in vitro of Pneumocystis carinii with macrophages and L-cells. J Exp Med 1978;147:157–170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Hidalgo HA, Helmke RJ, German VF, Mangos JA. Pneumocystis carinii induces an oxidative burst in alveolar macrophages. Infect Immun 1992;60:1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Laursen AL, Møller B, Rungby J, Petersen CM, Andersen PL. Pneumocystis carinii–induced activation of the respiratory burst in human monocytes and macrophages. Clin Exp Immunol 1994;98:196–202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Lasbury ME, Durant PJ, Bartlett MS, Smith JW, Lee CH. Correlation of organism burden and alveolar macrophage counts during infection with Pneumocystis carinii and recovery. Clin Diagn Lab Immunol 2003;10:293–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Lasbury ME, Tang X, Durant PJ, Lee CH. Effect of transcription factor GATA-2 on phagocytic activity of alveolar macrophages from Pneumocystis carinii–infected hosts. Infect Immun 2003;71:4943–4952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lasbury ME, Merali S, Durant PJ, Tschang D, Ray CA, Lee CH. Polyamine-mediated apoptosis of alveolar macrophages during Pneumocystis pneumonia. J Biol Chem 2007;282:11009–11020. [DOI] [PubMed] [Google Scholar]
- 10.Farley KS, Wang LF, Razavi HM, Law C, Rohan M, McCormack DG, Mehta S. Effects of macrophage inducible nitric oxide synthase in murine septic lung injury. Am J Physiol Lung Cell Mol Physiol 2006;290:L1164–L1172. [DOI] [PubMed] [Google Scholar]
- 11.Bogdan C. Nitric oxide and the immune response. Nat Immunol 2001;2:907–916. [DOI] [PubMed] [Google Scholar]
- 12.Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 1988;141:2407–2412. [PubMed] [Google Scholar]
- 13.Fleming SD, Iandolo JJ, Chapes SK. Murine macrophage activation by staphylococcal exotoxins. Infect Immun 1991;59:4049–4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Jun CD, Kim SH, Soh CT, Kang SS, Chung HT. Nitric oxide mediates the toxoplasmastatic activity of murine microglial cells in vitro. Immunol Invest 1993;22:487–501. [DOI] [PubMed] [Google Scholar]
- 15.Liew FY, Li Y, Millott S. Tumour necrosis factor (TNF-alpha) in leishmaniasis: II. TNF-alpha–induced macrophage leishmanicidal activity is mediated by nitric oxide from L-arginine. Immunology 1990;71:556–559. [PMC free article] [PubMed] [Google Scholar]
- 16.Lee SC, Kress Y, Dickson DW, Casadevall A. Human microglia mediate anti–Cryptococcus neoformans activity in the presence of specific antibody. J Neuroimmunol 1995;62:43–52. [DOI] [PubMed] [Google Scholar]
- 17.Ke X, Terashima M, Nariai Y, Nakashima Y, Nabika T, Tanigawa Y. Nitric oxide regulates actin reorganization through cGMP and Ca(2+)/calmodulin in Raw 264.7 cells. Biochim Biophys Acta 2001;1539:101–113. [DOI] [PubMed] [Google Scholar]
- 18.Lasbury ME, Durant PJ, Liao CP, Lee CH. Effects of decreased calmodulin protein on the survival mechanisms of alveolar macrophages during Pneumocystis pneumonia. Infect Immun 2009;77:3344–3354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Sherman MP, Loro ML, Wong VZ, Tashkin DP. Cytokine- and Pneumocystis carinii–induced L-arginine oxidation by murine and human pulmonary alveolar macrophages. J Protozool 1991;38:234S–236S. [PubMed] [Google Scholar]
- 20.Downing JF, Kachel DL, Pasula R, Martin WJ II. Gamma interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine– and tumor necrosis factor–dependent mechanisms. Infect Immun 1999;67:1347–1352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ishimine T, Kawakami K, Nakamoto A, Saito A. Analysis of cellular response and gamma interferon synthesis in bronchoalveolar lavage fluid and lung homogenate of mice infected with Pneumocystis carinii. Microbiol Immunol 1995;39:49–58. [DOI] [PubMed] [Google Scholar]
- 22.Hanano R, Reifenberg K, Kaufmann SH. Activated pulmonary macrophages are insufficient for resistance against Pneumocystis carinii. Infect Immun 1998;66:305–314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Shellito JE, Kolls JK, Olariu R, Beck JM. Nitric oxide and host defense against Pneumocystis carinii infection in a mouse model. J Infect Dis 1996;173:432–439. [DOI] [PubMed] [Google Scholar]
- 24.Queener SF, Bartlett MS, Nasr M, Smith JW. 8-aminoquinolines effective against Pneumocystis carinii in vitro and in vivo. Antimicrob Agents Chemother 1993;37:2166–2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kolls JK, Habetz S, Shean MK, Vazquez C, Brown JA, Lei D, Schwarzenberger P, Ye P, Nelson S, Summer WR, et al. IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells. J Immunol 1999;162:2890–2894. [PubMed] [Google Scholar]
- 26.Lasbury ME, Lin P, Tschang D, Durant PJ, Lee CH. Effect of bronchoalveolar lavage fluid from Pneumocystis carinii–infected hosts on phagocytic activity of alveolar macrophages. Infect Immun 2004;72:2140–2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Worsham PL, Goldman WE. Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores. J Med Vet Mycol 1988;26:137–143. [PubMed] [Google Scholar]
- 28.Wang SH, Zhang C, Lasbury ME, Liao CP, Durant PJ, Tschang D, Lee CH. Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia. Microbes Infect 2008;10:334–341. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Zhang C, Wang SH, Lasbury ME, Tschang D, Liao CP, Durant PJ, Lee CH. Toll-like receptor 2 mediates alveolar macrophage response to Pneumocystis murina. Infect Immun 2006;74:1857–1864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Liao CP, Lasbury ME, Wang SH, Zhang C, Durant PJ, Murakami Y, Matsufuji S, Lee CH. Pneumocystis mediates overexpression of antizyme inhibitor resulting in increased polyamine levels and apoptosis in alveolar macrophages. J Biol Chem 2009;284:8174–8184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Eissa NT, Yuan JW, Haggerty CM, Choo EK, Palmer CD, Moss J. Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization. Proc Natl Acad Sci USA 1998;95:7625–7630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophoton Int 2004;11:36–42. [Google Scholar]
- 33.Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE, Byrns RE. Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine. Proc Natl Acad Sci USA 1993;90:8103–8107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Castillo L, deRojas TC, Chapman TE, Vogt J, Burke JF, Tannenbaum SR, Young VR. Splanchnic metabolism of dietary arginine in relation to nitric oxide synthesis in normal adult man. Proc Natl Acad Sci USA 1993;90:193–197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Stuehr DJ, Marletta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci USA 1985;82:7738–7742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Perrella MA, Yoshizumi M, Fen Z, Tsai JC, Hsieh CM, Kourembanas S, Lee ME. Transforming growth factor-β1, but not dexamethasone, down-regulates nitric-oxide synthase mRNA after its induction by interleukin-1β in rat smooth muscle cells. J Biol Chem 1994;269:14595–14600. [PubMed] [Google Scholar]
- 37.Blasko E, Glaser CB, Devlin JJ, Xia W, Feldman RI, Polokoff MA, Phillips GB, Whitlow M, Auld DS, McMillan K, et al. Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors. J Biol Chem 2002;277:295–302. [DOI] [PubMed] [Google Scholar]
- 38.Watts RN, Ponka P, Richardson DR. Effects of nitrogen monoxide and carbon monoxide on molecular and cellular iron metabolism: mirror-image effector molecules that target iron. Biochem J 2003;369:429–440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Taylor BS, Alarcon LH, Billiar TR. Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Mosc) 1998;63:766–781. [PubMed] [Google Scholar]
- 40.Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001;357:593–615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Panda K, Rosenfeld RJ, Ghosh S, Meade AL, Getzoff ED, Stuehr DJ. Distinct dimer interaction and regulation in nitric-oxide synthase Types I, II, and III. J Biol Chem 2002;277:31020–31030. [DOI] [PubMed] [Google Scholar]
- 42.Stuehr DJ, Kwon NS, Nathan CF, Griffith OW, Feldman PL, Wiseman J. N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine. J Biol Chem 1991;266:6259–6263. [PubMed] [Google Scholar]
- 43.Mayer B, Hemmens B. Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem Sci 1997;22:477–481. [DOI] [PubMed] [Google Scholar]
- 44.López A, Bernardo B, López-Herce J, Cristina AI, Carrillo A. Methaemoglobinaemia secondary to treatment with trimethoprim and sulphamethoxazole associated with inhaled nitric oxide. Acta Paediatr 1999;88:915–916. [DOI] [PubMed] [Google Scholar]
- 45.Buratti T, Joannidis M, Pechlaner C, Wiedermann CJ. Systemic hypotension on withdrawal from inhaled nitric oxide in an adult patient with acute respiratory distress syndrome. Crit Care Med 1999;27:441. [DOI] [PubMed] [Google Scholar]