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IMMUNOLOGY, HEALTH, AND DISEASE |
Department of Poultry Science, University of Arkansas, Fayetteville 72701
1 Corresponding author: otbowen{at}uark.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-nitro-L-arginine methyl ester was injected to inhibit ongoing NO production. In Experiment 1, the amplitude of the peak and postpeak PH responses to LPS were correlated with the quantity of NO produced by LPS-stimulated cultured PMNC from broilers in the control group but not for MP survivors. In Experiment 2, the postpeak PH response to LPS was correlated with the quantity of NO produced by LPS-stimulated PMNC from broilers in the relaxed line, but not in the susceptible or resistant lines. In all groups, N-nitro-L-arginine methyl ester injections triggered substantial increases in pulmonary arterial pressure (
8 mm Hg), thereby revealing a significant ongoing modulation by NO of the PH response to LPS. We concluded that most of the modulatory NO generated in vivo during the acute PH response to LPS (within 60 min postinjection) likely is produced by constitutive NO synthase in the vascular endothelium. In addition, the NO produced by inducible NO synthase in PMNC appeared to have modulated the LPS-stimulated PH responses of unselected broilers having the broadest range of pulmonary vascular capacities (control broilers and relaxed line), but not in broilers whose pulmonary vascular capacities had been selected to represent the higher (MP survivors, resistant line) or lower (susceptible line) extremes of the population.
Key Words: broiler • pulmonary hypertensive responsiveness • lipopolysaccharide • nitric oxide • mononuclear cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Variability among individuals in the amplitude and duration of their PH responses to LPS may reflect anatomical differences in the numbers of pulmonary vascular channels, in the responsiveness of the pulmonary vascular endothelium or smooth muscle, or in the proportions of vasoactive mediators produced during the inflammatory cascade triggered by LPS. Current evidence supports the hypothesis that LPS-mediated PH reflects an innate inflammatory response in which a platelet-activating factor triggers the release of vasoconstrictors (thromboxane and serotonin) from thrombocytes. Concurrently, LPS stimulates the synthesis of the potent pulmonary vasodilator nitric oxide (NO), which modulates (attenuates) the PH response to vasoconstrictors (Wideman et al., 2001, 2004; Wideman and Chapman, 2004; Chapman et al., 2005; Chapman and Wideman, 2005).
The synthesis of NO is catalyzed by inducible NO synthase (iNOS; NOS-2), which is irreversibly activated to produce high concentrations of NO, and by endothelial NO synthase (eNOS; NOS-3), which produces transient bursts of NO at low but physiologically effective concentrations (Albrecht et al., 2003). Lipopolysaccharide and inflammatory cytokines stimulate iNOS expression and increase the activity of eNOS isoforms in numerous pulmonary cells, including alveolar macrophages in mammals (Fujii et al., 1998). Macrophages derived from chickens respond to LPS stimulation in vitro by expressing iNOS and producing copious quantities of NO (Hussain and Qureshi, 1997). Lipopolysaccharide-mediated NO synthesis by broiler peripheral blood mononuclear cells (PMNC; monocytes, thrombocytes, and lymphocytes) in vitro can be inhibited by the specific iNOS inhibitor aminoguanidine and by N-nitro-L-arginine methyl ester (L-NAME), which is virtually equipotent as a competitive inhibitor of both iNOS and eNOS (Wideman et al., 2006).
One objective of the present study was to evaluate possible correlations between LPS-stimulated NO synthesis by PMNC and the amplitude of the PH response to LPS. We tested the hypothesis that broilers whose PMNC respond to LPS by synthesizing large amounts of NO in vitro would be expected to exhibit attenuated PH responses to LPS in vivo. If inverse correlations of this type can be confirmed, then the responses of PMNC obtained by blood sampling broilers theoretically could be used to predict key performance characteristics of those individuals, including the impact of LPS on their pulmonary vascular reactivity. Therefore, blood samples were collected for PMNC isolation and in vitro stimulation with LPS, then the same broilers were anesthetized, and their PH responses to LPS were evaluated.
Nitric oxide also modulates the PH response to pulmonary microvascular obstruction caused by i.v. injections of cellulose microparticles. Intravenous microparticle injections are used to eliminate from broiler populations those individuals having the most restrictive pulmonary vascular capacity (Wideman and Erf, 2002; Wideman et al., 2003). Microparticles entrapped within the pulmonary vasculature initiate the formation of focal aggregates of thrombocytes and PMNC within 20 min postinjection, thereby establishing the inflammatory milieu for the contemporaneous release of vasoconstrictors by thrombocytes, and NO synthesized by iNOS in activated PMNC (Wideman and Erf, 2002; Wang et al., 2003). Additional NO also can be synthesized by eNOS when microparticle entrapment causes increased shear stress (due to increased rates of blood flow) to be exerted on endothelial cells lining unobstructed pulmonary vascular channels (Wideman et al., 2005). Administering L-NAME to block NO synthesis prior to injecting microparticles revealed PH responses that were substantially greater in amplitude and more prolonged in duration when compared with those of control broilers that had not been pretreated with L-NAME. In addition, the mortality triggered by i.v. microparticle injections more than doubled when L-NAME was combined with microparticle injection doses that otherwise caused relatively low mortality in the absence of L-NAME. These demonstrations that NO-mediated dilation of the pulmonary vasculature can partially attenuate microparticle-induced increases in pulmonary vascular resistance raise the possibility that broiler survivors of microparticle injections may possess favorable inflammatory response profiles (e.g., high rates of NO production, low rates of vasoconstrictor production, or both) in addition to having an anatomically robust pulmonary vascular capacity (Wideman et al., 2004, 2005). Accordingly, a second objective of the present study was to compare the PAP responses to LPS and L-NAME in male broilers from a base population (control group) and in flock mates that were sufficiently robust to have survived a 60% lethal dose of an i.v. injection of cellulose microparticles (MP survivor group). If an innately favorable inflammatory response profile at least partially supports survival after a 60% lethal dose of a microparticle injection, then the MP survivors in Experiment 1 ought to exhibit lower amplitude increases in PAP following an i.v. LPS injection (enhanced modulation by NO) or larger amplitude increases in PAP following a subsequent injection of L-NAME (exposure of unmodulated LPS-mediated vasoconstriction).
In Experiment 2, the in vitro and in vivo responses to LPS were evaluated in broilers from a relaxed (control) line, as well as in broilers selected for 10 generations under conditions of hypobaric hypoxia for susceptibility (susceptible line) or resistance (resistant line) to PHS. The relaxed line was the base population from which the susceptible and resistant lines had been selected (Anthony et al., 2001; Balog, 2003). Our objectives were to determine whether these lines had diverged in their responsiveness to LPS and to assess possible correlations between LPS-stimulated NO synthesis by PMNC in vitro and the amplitude of the PH response to LPS in vivo. If selection for susceptibility or resistance to PHS involved coselection for LPS response profiles, then we would expect susceptible broilers’ PMNC to produce less NO in vitro when compared with PMNC from resistant broilers, and the amplitude of the PH response to LPS in vivo should be higher in susceptible than in resistant broilers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male broilers were transported from a commercial hatchery (Cobb-Vantress Inc., Fayetteville, AR) to the Poultry Environmental Research Lab at the University of Arkansas Poultry Research Farm on September 22, 2004 (d 1). They were placed on wood shavings litter in environmentally controlled chambers. The temperature was kept at 33°C during d 1 to 5, 29°C for d 6 to 10, 26°C during d 11 to 16, and 24°C thereafter. Broilers were exposed to 24 h of light through d 5, and, subsequently, were kept at 16L:8D. They had ad libitum access to feed and water.
On d 19, microgranular carboxymethyl-32 ion exchange cellulose (Fisher Scientific, St. Louis, MO) suspended at 0.02 g/mL in heparinized saline [150 units ammonium heparin (Sigma Chemical Co., St. Louis, MO)/mL of 0.9% NaCl] was injected into the basilic vein (wing vein) of 55 broilers. Injection was carried out using a 1-mL tuberculin syringe with a 22-gauge needle. The injection dose needed to trigger approximately 60% mortality within 24 h post-injection was determined by injecting broilers with 0.5 (n = 11), 0.6 (n = 22), or 0.7 mL (n = 22) of the cellulose microparticle suspension. On d 20, 0.6 mL of the 0.02 g/mL cellulose microparticle suspension was injected into 436 broilers, leading to 60% (260/436) mortality within 24 h postinjection. Control broilers were not injected with microparticles or heparinized saline. All birds were wing-banded according to their control or MP survivor grouping. Immune-mediated clearance of entrapped cellulose microparticles restores patency to the pulmonary vasculature of surviving birds within 18 d postinjection, at which time the intrapulmonary inflammatory response also has subsided (Wideman et al., 2002). On d 41, blood samples were collected from 21 control and 20 MP survivor broilers. Three-milliliter blood samples were taken from the right wing vein using heparinized syringes fitted with 23-gauge needles. One milliliter of the blood was used to determine blood leukocyte concentrations with the Abbott Diagnostics Cell Dyn 3500 (Abbott Diagnostics, Abbott Park, IL) automated hematology analyzer and for blood smears. Blood smears were stained using Wright’s stain adjusted for avian blood staining (Lucas and Jamroz, 1961). The remaining 2 mL of blood was used for isolation of PMNC.
Experiment 2: Broiler Management
Strategies for selecting ascites susceptible and resistant broiler lines under conditions of hypobaric hypoxia have been described previously (Anthony et al., 2001; Balog, 2003). After the eighth generation of selection, progeny of the susceptible and resistant lines exhibited ascites mortalities of 98.6 and 26.0%, respectively, when grown under hypobaric conditions (Pavlidis, 2003). Male progeny from the 10th generation of these lines, as well as progeny from the relaxed line, were transported on the day of hatch (December 2, 2004; d 1) to the Poultry Environmental Research Lab at the University of Arkansas Poultry Research Farm and were reared as previously described in Experiment 1. On d 34, 3-mL blood samples were collected from 12 susceptible, 11 relaxed, and 11 resistant broilers and dispersed for blood leukocyte analysis and PMNC isolation as for Experiment 1.
Blood Leukocyte Proportions and Concentrations
An automated hematology analyzer (Abbott Diagnostics) calibrated for chicken blood was used to determine concentrations of white blood cells (WBC; 103/µL), red blood cells (RBC; 106/µL), and thrombocytes (103/µL). Based on our experience, the proportions among various chicken blood leukocytes (lymphocytes, heterophils, monocytes, basophils, and eosinophils) are not reliably estimated by this instrument. Hence, the proportions among blood leukocytes were determined manually using blood smears as previously described in Wang et al. (2003). Blood smears were stained with Wright’s stain (Lucas and Jamroz, 1961) and examined using oil immersion (1,000x magnification) using a bright field microscope.
PMNC Isolation
To validate the reproducibility of LPS-stimulated NO production in PMNC cultures from individual birds, a pilot study was conducted in which blood samples were drawn from the same birds every 3 d over the course of a week. The PMNC suspensions (3 samples from 8 broilers) as well as those prepared in Experiments 1 and 2 were isolated by density gradient centrifugation over Ficoll 1077 (Sigma Chemical Co.; Wideman et al., 2006).
Nitrite Assay
Nitrite assay using Griess reagent [1:1 ratio of 1% sulfanilamide (Sigma Chemical Co.) with phosphoric acid to 0.1% N-1-napthylethylenediamine dihydrochloride (Sigma Chemical Co.)] was used to determine NO production by PMNC that were cultured for 24 h with or without LPS (Sigma Chemical Co.; 20 µg from S. typhimurium). For each culture, the average absorbance of triplicate estimates was calculated, and the corresponding nitrite concentration (micromolar) was determined using the standard curve equation describing the linear relationship between absorbance and nitrite concentration (Wideman et al., 2006).
Detection of NO-Producing Cells by Flow Cytometry
To determine the type(s) of cells within the PMNC cultures that participate in the production of NO and to examine the relative amounts of NO produced by each cell, PMNC suspensions were examined by flow cytometry. Two hundred microliters of PMNC at 1 x 107 cells/mL was placed in sterile siliconized centrifuge tubes with 10 µL of 2 mg/mL LPS [from S. typhimurium (Sigma Chemical Co.); LPS-stimulated cultures] or 10 µL of PBS (unstimulated) and incubated in a humidified incubator for 24 h at 40°C with 5% CO2. After incubation, 300 µL of complete culture medium was added to each culture. To detect NO production, 2 µM 4-amino-5-methylamino-2',7'-difluorescein (DAF-FM) diacetate (Molecular Probes Inc., Eugene, OR) was added to the cell suspension in each tube, and cultures were maintained at room temperature for 20 min. The DAF-FM diacetate passively diffuses across the cell membrane and reacts with esterases present in the cell to form DAF-FM. The reaction between DAF-FM and NO generates fluorescence and, hence, individual NO-producing cells can be detected by flow cytometry. After incubation, 8 mL of cold PBS was added to the cell suspension in each tube, and cells were centrifuged at 250 x g at 4°C for 8 min. The supernatant fluid was discarded, and the pellet was resuspended in 0.5 mL of PBS. The cells were then incubated without direct light for 15 min at room temperature. Each cell suspension was transferred into 5-mL Falcon tubes (Becton, Dickinson and Co., San Jose, CA) for fluorescence analysis using a Becton Dickinson FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a 488-nm argon laser. Cell population analysis was conducted using the CellQuest software (Henry Schein Inc., Melville, NY). For analysis, a dot plot representing forward scatter (FSC; size) vs. side scatter (SSC; internal complexity) characteristics was generated. A region was then drawn around the live cell population. For chicken PMNC, this region contains a population of small cells with relatively low SSC characteristics (thrombocytes and lymphocytes) and a large cell population, also with relatively low SSC levels (monocytes). The FSC and SSC characteristics of live and dead cells were previously determined using propidium iodide staining. Moreover, the phenotype(s) of the small and the large cell population was determined based on staining patterns following indirect immunofluorescent staining with K1 (hybridoma culture supernates, gift from H. S. Lillehoj, USDA-ARS, Beltsville, MD; Oláh et al., 2001; specific for thrombocytes and monocytes), K55 (hybridoma culture supernatants, gift from H. S. Lillehoj, USDA-ARS; Chung et al., 1991; specific for lymphocytes and monocytes) monoclonal antibodies (data not shown), and direct immunofluorescent staining using the KUL01 mouse anti-chicken monocyte-and macrophage-specific monoclonal antibody (SouthernBiotech; Wigley et al., 2001). Using this approach, the large PMNC population was confirmed to be the monocyte population. To focus analysis on the monocyte population, another region was also drawn around the FSC-SSC display of the large PMNC population (monocytes). To examine fluorescence characteristics of the PMNC populations, a dot-plot FSC vs. relative fluorescence intensity at 515 to 545 nm (FL-1) was generated with cells gated for either region. Although cell populations without DAF-FM diacetate treatment were included to set up the flow cytometer for acquisition, due to the high background staining of DAF-FM diacetate of all cells, this was not found to be a useful guide to set up the cutoff between NO-producing cells and background fluorescence. Hence, for cell population analyses, the cutoff between fluorescence-positive and fluorescence-negative PMNC was set based on comparison of FL-1 characteristics from unstimulated and LPS-stimulated cultures that were incubated with DAF-FM diacetate. Positive fluorescence was consistently detected in the large PMNC population (monocytes) and not in the small PMNC population (thrombocytes and lymphocytes). A typical dot-plot display of NO-production-related fluoresence by monocytes (second region gate) from an unstimulated PMNC culture and its LPS-stimulated counterpart is shown in Figure 1
. Data were reported as the percentage of monocytes in the PMNC suspension, the percentage of monocytes that produce NO (fluorescence-positive), and the mean fluorescence intensity of NO-producing monocytes.
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The same broilers from which blood samples previously had been collected for leukocyte analyses and PMNC culture were anesthetized to a surgical plane at 43 to 51 d of age for control broilers (3,332 ± 45 g of BW, mean ± SEM) and MP survivors (3,275 ± 61 g of BW) in Experiment 1 and at 34 to 38 d of age for relaxed (1,975 ± 77 g of BW), susceptible (1,998 ± 36 g of BW), and resistant (1,924 ± 43 g of BW) broilers in Experiment 2. Ketamine HCl (40 mg/kg of BW; Henry Schein Inc.) and allobarbital [5,5-diallybarbituric acid (Fisher Scientific, St. Louis, MO); 16 mg/kg BW] were injected intramuscularly, and the birds were secured in dorsal recumbency on a surgical board. A wing vein was cannulated for i.v. injections, and the pulmonary artery was cannulated to record the PAP, as described previously (Wideman et al., 2006).
The baseline (control) PAP was recorded for 10 min, after which 1 mg of LPS (0.5 mL of 2 mg/mL of LPS from S. typhimurium) was injected i.v. This 1-mg dose of LPS has been shown to elicit maximal PH responses in broilers of similar age and BW range (Wideman et al., 2001; Wang et al., 2002b, 2003; Chapman et al., 2005). The PAP was monitored for 40 min post-LPS injection, after which 100 mg of L-NAME (Cayman Chemical Co.; 100 mg/mL) was injected i.v. It is known that L-NAME is virtually equipotent as a competitive inhibitor of both iNOS and eNOS, and doses of 10 to 50 mg of L-NAME/kg of BW previously were shown to significantly amplify and prolong the PH responses of broilers to LPS and microparticle injections (Wideman and Chapman, 2004; Bowen et al., 2006; Wideman et al., 2006). The PAP was monitored for 10 min after the L-NAME injection. The experiment was terminated with 10-mL i.v. injection of 0.1 M KCl. The heart was dissected to obtain the right ventricular to total ventricular weight (RV:TV) ratio as an index of right ventricular work and PAP.
The PAP responses were analyzed at predetermined time points over a 60-min interval, including at the start of PAP recording, immediately before injecting LPS, 5 min post-LPS, 10 min post-LPS, 15 min post-LPS, at the peak PAP response to LPS, 30 min post-LPS, 40 min post-LPS, and at the peak PAP after L-NAME was injected.
Data Analysis and Statistics
Correlation comparisons were conducted using the nitrite assay data for in vitro NO production (micromolar) by LPS-stimulated PMNC cultures as the independent variable and the net change in PAP amplitude after injecting LPS in vivo as the dependent variable. For each broiler, 3 different PAP amplitudes were separately correlated with PMNC NO production, as a means of evaluating the possible modulatory impact of NO during separate phases of the in vivo PH response to LPS. The peak PAP amplitude was calculated as the difference between PAP immediately before injecting LPS and the maximal peak PAP achieved in response to LPS. The postpeak PAP amplitude was calculated as the difference between the maximal peak PAP response to LPS and the PAP at 40 min post-LPS injection. The L-NAME amplitude was calculated as the difference between the PAP at 40 min post-LPS injection and the PAP plateau reached within 5 min after inhibiting NOS with L-NAME. These amplitudes bracket all phases of the acute PH response to LPS during which NO may have exerted a modulatory influence.
The SYSTAT (Systat Software Inc., Point Richmond, CA ) ANOVA was used to compare differences among groups within an experiment for blood leukocyte concentrations and proportions, NO production based on nitrite assay, and data obtained based on flow cytometric analysis. The PMNC collected repeatedly from individual broilers were analyzed by ranking each broiler’s performance within the group for each of the 3 d of sample collection. The lowest NO producer was ranked as a 1, whereas the highest NO producer was ranked as an 8. The ranks for each broiler over 3 PMNC isolation periods were averaged, and the CV was obtained for each broiler. The CV values for the 4 highest- and 4 lowest-ranked NO producers were compared using SYSTAT t-test. Production of NO and flow cytometry data were also analyzed using SYSTAT 2-way ANOVA to determine the main effect of LPS treatment (diluent vs. LPS) and type of broiler (control vs. MP survivors, Experiment 1; relaxed, susceptible, and resistant lines, Experiment 2) and their interactions. Differences in PAP within a group over the 60-min interval were determined using Sigma Stat (Jandel Scientific, San Rafael, CA) repeated measures ANOVA and the Tukey test or Dunnett’s method. The PAP differences among groups within each time interval were evaluated using Sigma Stat 1-way ANOVA with the Tukey test, or the Kruskal-Wallis 1-way ANOVA on ranks. Differences in RV:TV ratios were analyzed using Sigma Stat 1-way ANOVA. Correlation values were obtained using Sigma Stat linear regression and Pearson product moment correlation analysis. The PAP were analyzed by groups to determine whether there was a treatment effect.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PMNC cultures were prepared by repeatedly blood sampling the same birds at 3-d intervals. Concentrations of NO detected in the culture medium exhibited reasonable intraindividual consistency, although wide variations in NO concentrations were observed among different individuals. The mean NO concentration for the 4 broilers whose PMNC cultures consistently produced the most NO differed from the NO concentrations of the 4 broilers whose PMNC cultures consistently produced the least NO (highest NO concentration = 51.3 µM; lowest NO concentration = 19.6 µM; P = 0.049). The consecutive concentrations for the highest NO-producing individuals averaged (means ± SEM) 46.7 ± 9.6, 35.1 ± 7.3, and 32.5 ± 8.7 µM on d 1, 3, and 6, respectively. The consecutive concentrations of NO for the lowest NO-producing individuals averaged (means ± SEM) 35.4 ± 1.5, 14.8 ± 0.7, and 11.7 ± 2.4 µM on d 1, 3, and 6, respectively. However, the CV for the high-NO producers did not differ from the CV for the low-NO producers (P = 0.151), demonstrating similar intraindividual variation across 3 separate sample collections, regardless of the relative ranking for total 24-h NO production by PMNC cultures.
Experiment 1
Blood Leukocyte Analyses.
There were no differences in the concentrations of WBC, RBC, or thrombocytes when control broilers were compared with MP survivors (Table 1). There also were no differences in the percentages of lymphocytes, heterophils, monocytes, eosinophils, and basophils between control broilers and MP survivors.
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. The PMNC that had not been stimulated with LPS did not produce consistently detectable levels of NO, whereas PMNC cultures stimulated with LPS produced higher (P < 0.001) levels of NO that varied widely among individuals and averaged 29.6 ± 3.2 µM in control broilers and 29.5 ± 2.3 µM in MP survivors (P > 0.1 for group comparisons). The NO levels produced in vitro by LPS-stimulated PMNC cultures from broilers in the control group exhibited a total variation of 68 µM (range: 11.5 to 79.4 µM), whereas the total variation for the MP survivor group was 36 µM (range: 9.3 to 45.0 µM).
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). In unstimulated PMNC, cultures from control broilers, 28.2 ± 5.3% (mean ± SEM) of the monocytes produced NO, which did not differ from the 17.6 ± 3.0% of NO-producing monocytes in unstimulated cultures from MP survivors (P = 0.095). The percentages of NO-producing monocytes also did not differ (P = 0.185) when LPS-stimulated PMNC cultures were compared between control broilers (61.1 ± 6.7%; mean ± SEM) and MP survivors (49.8 ± 4.9%; mean ± SEM). In both groups, LPS did increase the percentages of NO-producing monocytes in PMNC cultures (P < 0.001). Furthermore, a significant group main effect demonstrated that PMNC cultures from the control group (unstimulated and LPS-stimulated combined) contained higher percentages of NO-producing monocytes than did the unstimulated and LPS-stimulated PMNC cultures from MP survivors (44.7 ± 3.6 vs. 33.7 ± 3.7%, respectively; P = 0.038). There were no interactions between groups (control and MP survivor) or treatments (unstimulated and LPS-stimulated). Comparing the variability among individual birds as reflected by the percentages of NO-producing monocytes in culture after LPS stimulation, a broad range existed among PMNC cultures from control broilers (range = 17.9 to 99.4%; mean = 61.1 ± 6.7%) and in cultures from MP survivors (range = 9.8 to 88.8%; mean = 49.8 ± 4.9%). The mean fluorescence intensities of fluorescence-positive monocytes, which are directly related to relative amounts of intracellular NO production by individual monocytes in the PMNC cultures, did not differ between control broilers and MP survivors within either the unstimulated or LPS-stimulated cultures. However, pooling the mean fluorescence intensity data across each LPS treatment of both the control and MP survivor groups demonstrated an LPS-stimulation main effect in which monocytes from unstimulated PMNC cultures were producing lower levels of intracellular NO than were monocytes from LPS-stimulated PMNC cultures (149.6 ± 20.0 vs. 356.1 ± 20.0, respectively; mean fluorescent intensity ± SEM, n = 41 for the combined PMNC cultures; P < 0.001). There were no interactions between groups and LPS treatments when observing the main effect differences.
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. The values for the control and MP survivor groups did not differ within any sample interval. The average baseline PAP for control broilers was 22.1 ± 1.2 mm Hg (range: 15.0 to 35.8 mm Hg), and for MP survivors the baseline PAP averaged 19.9 ± 0.8 mm Hg (range: 10.0 to 26.9 mm Hg). Injecting LPS caused an increase in PAP beginning approximately 15 min postinjection (P < 0.05), with the peak PH response occurring approximately 20 min postinjection. The LPS peak responses for control broilers averaged 39.9 ± 1.4 mm Hg, and MP survivors averaged 40.7 ± 1.4 mm Hg. After the PAP had subsided toward baseline pressure at approximately 40 min postinjection, at which time L-NAME was injected, and the PAP increased to a hypertensive plateau within 5 min (P < 0.05). The RV:TV ratios averaged 0.237 ± 0.01 and 0.235 ± 0.01 (mean ± SEM) for control broilers and MP survivors, respectively (P > 0.10).
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, panels A to C). The correlation using the peak PAP amplitude yielded a weakly predictive r2 and a significant inverse relationship with in vitro NO production by PMNC from control broilers (r2 = 0.195, P = 0.045) but not by PMNC from MP survivors (r2 = 0.035, P = 0.430) (Figure 4, panel A). The correlation using the postpeak PAP amplitude revealed a moderately predictive r2 and a significant inverse relationship with in vitro NO production by PMNC from control broilers (r2 = 0.406, P = 0.002) but not by PMNC from MP survivors (r2 = 0.000624, P = 0.917; Figure 4, panel B). The L-NAME PAP amplitudes were not correlated with in vitro NO production by PMNC from control broilers (r2 = 0.0462, P = 0.349) or MP survivors (r2 = 0.0982, P = 0.178; Figure 4, panel C).
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Blood Leukocyte Analyses.
There were no differences in the concentrations of WBC, RBC, or thrombocytes when broilers from the relaxed, susceptible, and resistant lines were compared (Table 3). Broilers from the relaxed line had higher concentrations of heterophils (2.8 ± 0.7 vs. 1.2 ± 0.2 x 103 cells/µL; mean ± SEM; P = 0.040) and monocytes (0.8 ± 0.2 vs. 0.5 ± 0.1 x 103 cells/µL; P = 0.043) than broilers from the resistant line. Relaxed broilers also had a higher percentage of heterophils when compared with resistant broilers (16.1 ± 2.3 vs. 9.8 ± 1.8%, respectively; P = 0.045). However, resistant broilers had a higher percentage of lymphocytes than broilers from the relaxed line (85.2 ± 2.2 vs. 76.7 ± 3.0%, respectively; mean ± SEM; P = 0.037). Susceptible broilers did not differ from the relaxed or resistant broilers within any category of leukocyte concentration or percentage.
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. Unstimulated PMNC cultures did not produce consistently detectable levels of NO, whereas LPS stimulation increased NO production in PMNC cultures from all groups (P < 0.001). The LPS-stimulated production of NO varied widely among individual PMNC cultures, but overall average values did not differ among the relaxed, susceptible, and resistant lines (34.4 ± 3.4, 32.4 ± 4.0, and 27.8 ± 4.2 µM, respectively; mean ± SEM; P = 0.492). The NO levels produced by LPS-stimulated PMNC cultures exhibited a net variation of 39 µM for the relaxed line (range: 16.2 to 55.4 µM), 46 µM for the susceptible line (range: 10.4 to 56.7 µM), and 41 µM for the resistant line (range: 6.3 to 47.5 µM).
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, resistant broilers had a greater percentage of monocytes in unstimulated PMNC cultures when compared with unstimulated PMNC cultures from relaxed broilers (8.4 ± 1.1 vs. 5.6 ± 0.7%, respectively; mean ± SEM; P = 0.029). The percentage of monocytes present in LPS-stimulated PMNC cultures did not differ among the relaxed, susceptible, and resistant lines (P = 0.083), although the percentages of NO-producing monocytes in LPS-stimulated PMNC cultures from individual birds within a group varied widely [relaxed (range = 10.4 to 53.7%; mean = 26.0 ± 3.3%), susceptible (range = 13.4 to 38.4%; mean = 24.2 ± 2.0%), and resistant (range = 4.0 to 36.6%; mean = 23.2 ± 2.7%) broilers]. Across all lines, LPS-stimulated cultures had higher percentages of monocytes than unstimulated cultures (main effect, P < 0.001). Unstimulated PMNC cultures from relaxed broilers contained a greater percentage of NO-producing monocytes than did unstimulated cultures from resistant broilers (17.1 ± 2.1 vs. 11.8 ± 1.6, respectively; mean ± SEM; P = 0.037), whereas the percentages of NO-producing monocytes in unstimulated cultures from susceptible broilers did not differ when compared with unstimulated cultures from relaxed and resistant broilers. Overall, LPS-stimulated PMNC cultures had higher percentages of NO-producing monocytes than did unstimulated PMNC cultures (24.5 ± 1.3 vs. 14.0 ± 1.3%, respectively; mean ± SEM; main effect, P < 0.001). The mean fluorescence intensities that are indicative of intracellular NO production by individual monocytes in PMNC culture did not differ among relaxed, susceptible, or resistant lines within either the unstimulated or LPS-stimulated categories. However, pooling the mean fluorescence intensity data, independent of broiler line, demonstrated that monocytes from LPS-stimulated PMNC cultures produced higher levels of NO than monocytes from unstimulated PMNC cultures (441.8 ± 27.0 vs. 262.4 ± 27.0 µM, respectively; mean ± SEM: main effect, P < 0.001; Table 4).
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. The initial baseline PAP for resistant broilers was lower than for susceptible broilers (20.4 ± 1.5, 25.7 ± 1.5, and 27.8 ± 2.6 mm Hg, respectively; mean ± SEM; P = 0.034). Resistant broilers maintained a lower PAP than susceptible broilers PAP through the 15 min post-LPS injection sample interval (P < 0.001). The LPS injection increased the PAP within approximately 20 min postinjection in all groups (peak sample interval, P = 0.05). The absolute magnitudes of the peak responses to LPS did not differ among lines, with susceptible broilers averaging 40.8 ± 1.4 mm Hg, relaxed broilers averaging 39.0 ± 1.4 mm Hg, and resistant broilers averaging 35.7 ± 2.6 mm Hg (P = 0.162). The resistant line again had PAP values that were lower than for the susceptible line at the 30-min (P = 0.025) and 40-min (P = 0.029) post-LPS injection sample intervals, whereas the PAP values for the relaxed line remained intermediate during these sample intervals. After the PH response to LPS had subsided to PAP values that did not differ from the initial baseline PAP for each line (40-min postinjection sample interval), injecting L-NAME i.v. caused PAP to increase to a hypertensive plateau within 5 min in all lines (P < 0.05). The absolute amplitude of the L-NAME-induced increase in PAP was lower in resistant broilers than in susceptible broilers (P = 0.028), but the L-NAME PAP plateau for relaxed broilers did not differ from either susceptible or resistant broilers (Figure 6). The RV:TV ratios averaged 0.246 ± 0.01, 0.232 ± 0.01, and 0.239 ± 0.01 (mean ± SEM) for relaxed, susceptible, and resistant broilers, respectively (P = 0.541).
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, panels A to C). The correlation using the postpeak PAP amplitude yielded a strong r2 value and a significant inverse relationship with in vitro NO production for relaxed broilers (r2 = 0.465, P = 0.021) but no correlation for susceptible broilers (r2 = 0.135, P = 0.240) or for resistant broilers (r2 = 0.010, P = 0.771; Figure 7, panel B). The correlation using the peak PAP amplitude and L-NAME amplitude did not reveal significant correlations (Figure 7, panels A and C; Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A primary objective was to evaluate possible correlations between LPS-stimulated NO synthesis by PMNC cultures and the amplitude of the PH response to LPS. In an attempt to obtain a range of PH amplitudes, we used broilers from control and MP survivor groups in Experiment 1 and from relaxed, susceptible, and resistant lines in Experiment 2. The baseline PAP values and PH responses to LPS observed in Experiment 1 replicated those previously reported by Chapman et al. (2005), who demonstrated that MP survivors had numerically lower baseline PAP values and numerically higher PH responses to LPS when compared with broilers from a control population. It is the progeny from MP survivor parents that have significantly lower baseline PAP values when compared with the progeny from unselected control parents (Wideman et al., 2006). The baseline PAP values and PH responses to LPS in Experiment 2 also replicated those previously reported by Bowen et al. (2006), who demonstrated that broilers from the resistant line have lower baseline PAP values and higher-amplitude PH responses to LPS when compared with broilers from the susceptible line. The higher-amplitude PH responses by resistant broilers did not translate into higher absolute peak PAP values when compared with the peak PAP attained by susceptible broilers (Figure 6
), which is consistent with repeated observations that blood regurgitates from the right ventricle back into the right atrium as the PAP approaches 40 mm Hg. Accordingly, broilers starting with an initially lower baseline PAP tend to exhibit higher amplitude PH responses before valvular regurgitation imposes an upper limit on the right ventricle’s capacity to further increase its pumping pressure (Wideman and Bottje, 1993; Wideman, 1999; Wideman et al., 2002, 2005, 2006; Chapman and Wideman, 2001; Bowen et al., 2006; Chapman et al., 2005). Evidently, the absolute threshold PAP at which valvular regurgitation occurs was not affected by 10 generations of selection for susceptibility or resistance to PHS (Figure 6), indicating that the fundamental difference between susceptible and resistant individuals resides in their pulmonary vascular capacity rather than in their cardiac contractility or valvular competence. Obviously, in some broilers, particularly those starting with an elevated baseline PAP, both valvular regurgitation truncated and NO synthesis modulated the peak PAP amplitude. The modulatory impact of NO was clearly evident from the very rapid (within 5 min) response to L-NAME, which restored PAP from baseline values back to hypertensive levels that approached the peak amplitude triggered by LPS. Previous studies have shown that L-NAME elicits only minor increases in pulmonary vascular resistance and PAP in broilers whose pulmonary vasculature has not been stimulated, whereas L-NAME profoundly increases both the amplitude and duration of PH responses to LPS and microparticle injections (Wang et al., 2002b; Weidong et al., 2002; Wideman and Chapman, 2004; Bowen et al., 2006; Wideman et al., 2005, 2006).
If broilers whose monocytes respond to LPS by synthesizing the highest amounts of NO in vitro consequently exhibit PH responses to LPS that are effectively modulated by NO in vivo, then we hypothesized that an inverse (negative) correlation would exist between the NO concentrations in culture medium from LPS-stimulated PMNC cultures and the amplitude of the PH response to LPS. Indeed, for the control group but not the MP survivor group in Experiment 1, the NO concentrations in medium from PMNC cultures were negatively correlated with both the peak PAP amplitude and the postpeak PAP amplitude. This negative correlation in the control group attained significance primarily due to data from 3 individuals, 2 of which had very high PAP amplitudes combined with very low NO concentrations, and 1 of which had the lowest PAP amplitude and the highest NO concentration (Figure 4, panel B). In Experiment 2, the correlation using the postpeak PAP amplitude again yielded a strong r2 value and a significant inverse relationship with in vitro NO production for broilers in the relaxed line but not for those in the susceptible and resistant lines. The existence of significant inverse correlations for broilers in the control group in Experiment 1 and the relaxed line in Experiment 2 suggests the in vitro responses of monocytes obtained from each broilers’ blood samples may be useful for predicting key in vivo performance characteristics, such as pulmonary vascular responsiveness to LPS, or may determine the PH response of that broiler should endotoxin become a problem.
Obviously, our approach to correlating in vitro and in vivo responses to LPS will require further experimentation and characterization before routine applications become a practical reality. For example, the evidence currently available consistently indicates that constitutive or eNOS primarily produces the NO that modulates the acute PH responses occurring within 60 min after i.v. LPS or microparticle injections, although some modulatory influence of NO produced by iNOS has been detected after administering the specific iNOS inhibitor aminoguanidine (Wideman and Chapman, 2004; Bowen et al., 2006; Wideman et al., 2005, 2006). In this context, it is relevant that a low percentage of the monocytes present in unstimulated PMNC cultures were producing NO, indicating that some variation probably exists in vivo in the basal levels of NO being produced by iNOS and released by monocytes into the circulation of broilers (Tables 2 and 4). The reversible activation of eNOS occurs transiently in response to increased shear stress exerted on the pulmonary vascular endothelium, whereas LPS-stimulated iNOS activation is considered essentially irreversible and may require several hours before maximal levels of enzyme expression and NO production are achieved (Stitt et al., 1997; Fujii et al., 1998; Pulido et al., 2000; Albrecht et al., 2003; Braulio et al., 2004; Chapman and Wideman, 2005). In the present study, we measured cumulative NO levels from PMNC cultures that were incubated for 24 h. The NO levels measured during this study reflect the maximum amount of NO that the PMNC were capable of producing over the course of 24 h, and a wide range of interindividual variation was demonstrated among all broiler lines evaluated. The NO measured from these PMNC cultures presumably was produced by iNOS, and full expression of iNOS by the monocytes in PMNC cultures may require more than 3 h after LPS stimulation before substantial differences in NO production can be measured (Wideman et al., 2006). Very low levels of NO that are essentially undetectable in the blood are capable of dilating the pulmonary vasculature (Weidong et al., 2002; Chapman and Wideman, 2005), therefore the earliest phases of iNOS activation in vivo theoretically can produce NO in sufficient quantities to dilate the pulmonary vasculature (Wideman et al., 2006). Nevertheless, the quantities of NO released into the PMNC culture medium over the course of 24 h may not correspond well with the NO being generated by iNOS during the acute PH response to LPS in the present study. In future studies, improved correlations between in vivo and in vitro values may be obtained by timing the comparisons to coincide with substantial NO production by iNOS (Bowen et al., 2006). The absence of any correlations between PMNC NO production and L-NAME amplitude may indicate that most of the NO produced during the time frame of the present study was derived from eNOS in response to increased shear stress exerted on the endothelium (Hampl and Herget, 2000).
An additional objective of the present study was to determine whether groups or lines of broilers known to differ in their susceptibilities to PHS also differed in their correlations between LPS-stimulated NO synthesis by PMNC in vitro and the amplitude of the PH response to LPS in vivo. If selection for susceptibility or resistance to PHS involved coselection for LPS response profiles, then we expected PHS-susceptible broilers’ PMNC to produce less NO in vitro when compared with PMNC from PHS-resistant broilers, and the amplitude of the PH response to LPS in vivo would be expected to be higher in PHS-susceptible than in PHS-resistant broilers. Significant inverse relationships between NO production from PMNC and postpeak PAP response to LPS were demonstrated in control (Experiment 1) and relaxed (Experiment 2) broilers but not in MP survivor, resistant, or susceptible broilers. Most susceptible broilers began with an elevated baseline PAP. Consequently, the peak response to LPS rapidly exceeded the threshold at which right ventricular regurgitation occurs. This physical truncation of the PAP peak amplitude in many susceptible broilers unavoidably helped to obscure the modulating effects of NO. However, in most resistant broilers, the peak PAP response to LPS was not truncated by valvular regurgitation. It is intriguing that selection for both PHS susceptibility (susceptible line) and resistance (MP survivors, resistant line) appears to have eliminated any relationship between LPS-stimulated NO production by PMNC cultures and the peak or postpeak PAP responses. No relevant trends in blood leukocyte concentrations or percentages were consistently observed, nor were wider ranges of total NO production by PMNC cultures observed in the relaxed line when compared with the susceptible or resistant lines. Accordingly, these observations are consistent with the hypothesis that the NO produced by LPS-stimulated PMNC was able to modulate the PAP in unselected broilers having the broadest range of pulmonary vascular capacities (control broilers and relaxed line) but not in broilers whose pulmonary vascular capacities had been concentrated toward the higher (MP survivors, resistant line) or lower (susceptible line) extremes of the population. Furthermore, variation in the PH response in vivo corresponded with the variable NO production by cultured PMNC, in that high NO producers in vitro had low PAP responses in vivo. Evidently, the anatomical or hemodynamic impact of the pulmonary vascular capacity overwhelmed the modulatory impact of monocyte-derived NO in the selected lines.
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ACKNOWLEDGMENTS |
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Received for publication September 19, 2005. Accepted for publication March 14, 2006.
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