Journal of Asthma, 43:95–99, 2006Copyright C© 2006 Taylor & Francis Group, LLCISSN: 0277-0903 print / 1532-4303 onlineDOI: 10.1080/02770900500497925

ORIGINAL ARTICLE

Exhaled Nitric Oxide in Asthma: Variability, Relation to Asthma Severity,and Peripheral Blood Lymphocyte Cytokine Expression

GOUTAM P. SHOME, M.D., PH.D.,1,∗ JOEL D. STARNES III, M.D., PH.D.,1 MICHAEL SHEARER, M.S.,2RONALD KENNEDY, PH.D.,2 ANTHONY WAY, M.D., PH.D., AHMED ARIF, M.D., PH.D.,

AND SHARMA PRABHAKAR, M.D.

1Division of Allergy & Immunology, Department of Internal Medicine, Texas Tech University, Health Sciences Center, Lubbock, Texas2Department of Microbiology & Immunology, Texas Tech University, Health Sciences Center, Lubbock, Texas

Exhaled nitric oxide has been used as a means of indirectly measuring the underlying inflammation in asthma. The objectives of the study were tomeasure exhaled nitric oxide levels in asthma patients and healthy volunteers, to study peripheral blood lymphocyte cytokine expression, and to studythe relationship between exhaled nitric oxide and intracellular cytokine expression. Exhaled nitric oxide was elevated in patients with moderate tosevere asthma and with treatment decreased in the first week reaching to a near normal level by 4 weeks. Elevated exhaled nitric oxide was associatedwith decreased IL-4 and IL-13 cytokine expression by CD8 lymphocytes.

Keywords exhaled nitric oxide, asthma, lymphocyte cytokine expression, inflammation, immune response

INTRODUCTION

Asthma is an inflammatory disease that involves activa-tion of host defense cells in the airways including T-helpercells, mast cells, eosinophils, and epithelial cells (1, 2). Asth-matic inflammation results in mucosal edema, mucous over-production, and bronchial constriction, all of which causeairway narrowing. Expert panel guidelines for asthma man-agement published by The National Heart, Lung and BloodInstitute suggest measuring airflow limitation as a markerfor airway caliber to evaluate and monitor asthma. For exam-ple, spirometry is recommended to assess airflow obstructionand response to bronchodilator therapy. However, spirometrydoes not measure inflammation, which may be present with-out airflow obstruction, and therefore other tests for responseto anti-inflammatory therapy are essential (3).

AIRWAY NITROGEN OXIDES

Noninvasive lung function tests have recently been devel-oped for airway inflammation. Most of these tests involvemeasurements of nitrogen oxides. In 1991, Gustafsson et al.(4) reported the presence of nitric oxide (NO) in humanbreath, and many investigators subsequently observed highlevels of nitric oxide in the breath of adults with asthma (5–10). NO is derived from molecular oxygen and the guani-dine nitrogen of L-arginine in a reaction catalyzed by NOsynthase (NOS); it is involved in a variety of biological

Supported in part by a seed grant from the Department of InternalMedicine, Texas Tech University Health Sciences Center.

∗Corresponding author: Goutam P. Shome, MD, PhD, 3601 4th Street,Division of Allergy & Immunology, Texas Tech University Health SciencesCenter, Lubbock, Texas 79430; E-mail: Goutam.shome@ttuhsc.edu

Abbreviations: eNO: Exhaled nitric oxide; IFN-γ : Interferon gamma;IL-4: Interleukin 4; IL-5: Interleukin 5; IL-13: Interleukin 13; NO: NitricOxide; NOS: Nitric oxide synthase; PBL: Peripheral blood lymphocytes;PBS: Phosphate buffered saline.

functions (11–18). Several critical nitrogen oxides are formedas a result of NOS activity in vivo, each with unique prop-erties and bioactivities. Well recognized of these is nitrite(NO−

2 ), which has potent antimicrobial properties and whenprotonated form nitrous acid (HNO2) (19). Peroxynitrite(OONO−), formed by reaction of NO with superoxide (O2),and nitrogen dioxide (NO2), formed by the reaction of NOwith oxygen, are both potentially cytotoxic species and havebeen implicated in the NOS-II mediated immune response,in oxidative lung injury, and in mutagenesis (20). Further,in vitro studies have demonstrated that NO can inhibit Th1cytokine secretion (21). Although NO has been implicated inthe immune response, little is known of the relationship be-tween exhaled NO (eNO) and Th1/Th2 cytokines expressionby peripheral blood lymphocytes in asthmatics and healthycontrols. The objectives of the study were to measure eNOlevels in asthma patients, and to compare them with healthyvolunteers without asthma; to assess the variability of eNO inpatients with moderate to severe asthma, and to compare thesedata with healthy volunteers and mild asthmatics; to studyintracellular cytokine (IL-4, IL-5, IL-13, and IFN-γ ) expres-sion in peripheral blood lymphocytes (PBL) in the asthmaticsand healthy controls; and to study the relationship betweeneNO and intracellular cytokine expression in vitro.

MATERIALS AND METHODS

Participant Selection and Baseline MeasurementsNineteen patients with asthma who presented to the Texas

Tech University Health Sciences Center Outpatient Clinicwere enrolled in the study. Seventeen age- and sex-matchedhealthy control subjects were enrolled from Health SciencesCenter staff. Healthy subjects were required to meet pre-determined exclusion criteria as outlined below. Informedconsents were obtained, and the study was approved by theInstitutional Review Board. Asthma diagnosis and severitywere assessed from symptoms, signs, and spirometry using

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96 G. P. SHOME ET AL.

the guidelines published by the National Heart, Lung andBlood Institute (22). In addition to these guidelines, an in-crease in the forced expiratory volume in 1 second (FEV1) ofat least 12% after treatment with 2.5 mg of albuterol sulfatewas required. Atopic status was assessed by skin prick test-ing with the following allergens: tree, grass, weed, dust mites,and cat dander (23). A wheal diameter of 3 mm or more withany of the two allergens was considered positive. Histamineand normal saline were used as positive and negative controls,respectively. Spirometry was performed by a trained individ-ual (Jaeger Masterscope Spirometer, Version 4.5, Hocchberg,Germany). Asthma patients were given pharmacologic ther-apy according to established guidelines (22). Records of treat-ment were made at each visit, and patients were required tovisit at the clinic at least once a week for 4 weeks. At eachvisit, detailed history, physical examination, spirometry, andeNO level were assessed.

Cell Collection and TreatmentTwenty milliliter (mL) of venous blood was drawn from

each participant into a Heparinized Vacutainer tube. The sam-ples were immediately centrifuged for 10 minutes at 4◦Cfor serum separation. Mononuclear cells were removed withHistopaque-1077 (Sigma Chemical Co, St. Louis, MO) byan established protocol. Samples were then stimulated withphorbol myristate acetate (40 ng/mL; Wako, Osaka, Japan)and ionomycin (2 µg/mL; Sigma Chemical Co, St Louis,MO) in the presence of brefeldin A (20 µg/mL) for 5 hoursat 37◦C in a humidified atmosphere of 5% CO2 in air (24).

Flow Cytometry AnalysisAliquots (1 × 106) of the stimulated cells were centrifuged

at 300 g for 5 minutes and resuspended in staining buffer toa volume of 0.1 mL. The cells were stained with a pretitratedamount (20 µL) of a FITC-conjugated mAb against the T-cell surface markers CD4 or CD8 for 20 minutes at roomtemperature. All staining antibodies, fixation, and permeabi-lization buffers were purchased from BD Biosciences, SanJose, CA, and used according to the manufactures instruc-tions. Cells were then centrifuged at 300 g for 5 minutes andwashed twice in phosphate buffered saline (PBS). Cells wereresuspended in fixation buffer and incubated for 20 minutesat room temperature. Cells were then centrifuged at 300 gfor 5 minutes and resuspended in permeabilizing solution for10 minutes at room temperature before staining with 20 µLof phycoerythrin-conjugated anti-IL-4, IL-5, IL-13, or IFN-γ for 20 minutes. Fluorescence intensity was measured witha FACS Vantange SE Cytometer. Approximately 1 × 105

cells were gated on the basis of forward and side light scatter.Isotype matched and labeled control subjects were used tomeasure background.

Clinical Measurements of eNOeNO was measured on-line by NO analyzer (CLD 88sp;

Eco Physics, Ann Arbor, MI) using the guideline publishedby the American Thoracic Society (23). This machine is sen-sitive to less than one part per billion. Study subjects beganby breathing NO free air from a filtering system, and thenexhaled against a low level of resistance (10 cm of water)without nose clips to close the velum and eliminate nasal con-

TABLE 1.—Baseline characteristics of asthma patients and healthy volunteers.

Control (n = 17) Mild (n = 11) Severe (n = 8)

Age 38.71 ± 13.04 ∗52.36 ± 17.10 38.25 ± 8.52Allergy Positive 4 (24%) 11 (100%) 8 (100%)FEV1 3.74 ± 1.01 2.97 ± 1.67 ∗2.41 ± 0.76FVC 4.53 ± 1.17 3.91 ± 1.74 3.57 ± 0.77FEV1/FVC 82.94 ± 9.48 ∗79.09 ± 10.74 ∗67.57 ± 15.87FEF25-75 4.1 ± 2.22 ∗2.774 ± 2.88 ∗1.76 ± 0.86

Data expressed as mean ± SEM; ∗ p < 0.05 vs. control.

tamination, into an open-ended reservoir at a fixed flow rate,with the plateau phase eNO recorded as the expired NO. Sincevalues are highly flow dependent, the same expiratory flowrate (0.05 ± 10% Ls/sec) was used for all measurements (25).

Exclusion CriteriaPatients and volunteers with the following conditions were

excluded: (1) chronic obstructive pulmonary disease, (2) cys-tic fibrosis, (3) lupus pneumonitis, and (4) sepsis, (5) respira-tory infection in the previous six weeks, (6) Congestive heartfailure, (7) History of smoking, and other systemic diseaseswith pulmonary symptoms (26–30).

Statistical AnalysisStatistical analysis was performed using statistical soft-

ware package (Sigmastat 3.1; Systat Software, Inc., PointRichmond, CA). Analysis of variance (ANOVA) with Holm-Sidak was used to compare eNO concentrations betweengroups. ANOVA was also used to determine correlations be-tween categorical variables and eNO concentrations. Univari-ate linear regression analysis was used to determine correla-tions between continuous variables and eNO concentrations.eNO values are reported as mean ± SEM in parts per billion.Two-sided tests yielding a p value of <0.05 were consideredstatistically significant.

RESULTS

Table 1 shows the baseline characteristics of asthma pa-tients and healthy volunteers. There was no significant ageor sex difference between the healthy controls and patientswith moderate to severe asthma. FEV1, FEV1/FVC, andFEF 25–75 were significantly lower in patients with mod-erate to severe asthma. All asthmatics enrolled in the studywere atopic. Figure 1 shows the eNO level in moderate

FIGURE 1.—Elevated baseline eNO in moderate to severe asthmatics when com-pared with healthy control subjects and mild asthmatics. ∗∗ p < 0.001. Data ex-pressed as mean ± SEM.

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EXHALED NITRIC OXIDE IN ASTHMA 97

FIGURE 2.—Variability of eNO in moderate to severe asthmatics when comparedto mild asthmatics and healthy control subjects. Data expressed as mean ± SEM.∗∗ p < 0.05 vs. control.

to severe asthmatics compared with mild asthmatics andhealthy control subjects. eNO was significantly elevated inmoderate to severe asthmatics compared to healthy controls(18.53 ± 2.00 ppb vs. 5.90 ± 0.90 ppb). eNO in mild asth-matics was not significantly different compared to healthycontrols (6.27 ± 3.79 ppb vs. 5.90 ± 3.7 ppb). There wasa gradual decline in eNO in moderate to severe asthmaticswith treatment; and by 4 weeks, eNO levels in moderate to se-vere asthmatics were not significantly different from the mildasthmatics and healthy controls (Figure 2). While there wasgradual decline in eNO level in asthmatics, FEV1 remainedessentially unchanged (Figure 3). Figure 4 demonstrates in-tracellular IFN-γ expression by peripheral blood CD4 andCD8 lymphocytes of patients with moderate to severe asthma,mild asthma, and healthy controls. IFN-γ expression, whilenot statistically significant, appears to be decreased in mod-erate to severe asthmatics. Figures 5A and 5B demonstratethe correlation between eNO with intracellular IL-4 & IL-13expression by CD8 lymphocytes of asthmatics and healthycontrol subjects. Elevated eNO was associated with decreasedIL-4 expression by CD8 positive lymphocytes (p < 0.02).Elevated eNO was also associated decreased IL-13 expres-sion in CD8 lymphocytes. A similar trend was also seen with

FIGURE 3.—In moderate to severe asthma, while FEV1 is unaltered over 4 weeksof therapy, the eNO level decreases significantly. Data expressed as mean ±SEM.

FIGURE 4.—In moderate to severe asthma, IFN-γ expression is decreased inboth CD4 and CD8 cells. Data expressed as mean ± SEM. Data not statisticallysignificant.

eNO and IL-4, eNO, and IFN-γ expression by CD4 lympho-cytes, but the results did not reach statistical significance.

DISCUSSION

Asthma is a chronic inflammatory disease of the airwaythat is treated with anti-inflammatory medications. However,in the routine clinical setting, we measure lung function

FIGURE 5.—Correlation between eNO and CD8 cell IL-4 (A) and IL-13(B) expression. Increased eNO levels are associated with diminished expres-sion of IL-4 and IL-13.

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98 G. P. SHOME ET AL.

rather than any inflammatory parameters. The gold stan-dard of assessment of airway inflammation is bronchoscopyand bronchial biopsy, but to perform regular biopsy in thischronic condition is inappropriate and unethical. This hasdriven the clinician scientist to look for other markers of air-way inflammation.

Multiple studies have established the fact that eNO is el-evated in patients with moderate to severe asthma (31–34).We have demonstrated that eNO level is significantly ele-vated in moderate to severe asthmatics and noted that eNOin mild asthmatics was no different from the healthy controlsubjects. There are few systematic observations of eNO vari-ability once asthma treatment has been initiated. We havefound that with initiation of therapy in moderate to severeasthmatics eNO starts declining in the first week; and by4 weeks, eNO in asthmatics is no different from healthy con-trol subjects. There is very little if any eNO variability inmild asthmatics and healthy control subjects. Our prelimi-nary observations suggest that decline in eNO in asthmaticswas similar irrespective of their differences in the combina-tions of anti-inflammatory therapies (inhaled corticosteroidsvs. inhaled corticosteroid plus long-acting bronchodilators).

Clinically it has been known for a while that NO inducesvasodilatation (35). Results of studies addressing the role in-haled NO on bronchial tone have been conflicting. Hogmanet al. (36) reported some bronchodilation if a reversible com-ponent in expiratory airflow limitation was present. Von Nied-ing et al. noticed 20% increases in airway resistance with anNO concentration above 20 ppm (37).

However, there is increasing evidence that NO mayplay a key role in nonspecific defense mechanisms againstpathogens, specifically, NO may be involved in the signalingbetween macrophages and T cells (38). It has been shown thatcloned murine Th1 cells, activated by antigens or mitogens,express inducible nitric oxide synthase (iNOS) and producesubstantial amounts of NO. Further, high concentrations ofNO inhibits Th1 cell proliferation and their production ofIL-2 and IFN-γ (21).

The relationship between eNO and intracellular cytokineexpression by PBL in asthmatics and healthy controls hasnot been studied previously. In the current study, we foundelevated eNO is associated with decreased IL-4 and IL-13expression by CD8 positive PBL. Similar negative correla-tion was also observed with IL-4 expression by CD4 positivelymphocytes, but the results were not statistically significant(data not shown). The decrease in Th2 cytokine expression(IL-4 and IL-13) by CD8-positive lymphocytes and its signifi-cant relation to eNO are interesting. There is a suggestion thata Th2 (IL-4 production) immune response to unrelated anti-gens can modify the functional potential of bystander-naı̈veCD8-positive lymphocytes, a mechanism that may explainthe viral-induced exacerbation of asthma (39). In vitro stud-ies in the mouse model have documented differentiation ofCD8+ lymphocytes in the presence of IL-4 to CD8+ lym-phocytes that produced IL-4 & IL-5 (40).

We have also observed significant decrease in IFN-γ ex-pression by the PBL of moderate to severe asthmatics, whichmay correlate with elevated eNO. Suppression of both Th1and Th2 cytokine in the peripheral blood lymphocytes (inasthmatics and healthy control subjects) in relation to ele-vated eNO level is a new and intriguing finding that needs

further study. To which extent this affects the asthma inflam-mation is unknown at this time. There are several limitationsof our study. Only adult asthmatics were enrolled, and not allpatients were steroid naı̈ve at the outset of the study. Corre-lation was sought between intracellular cytokine expressionby PBL and eNO, not peripheral blood NO. Cytokine ex-pression was studied in relatively small number patients withasthma (4) and healthy controls (6). All of our asthmaticswere atopic, whereas only 4 of 17 healthy volunteers wereatopic.

CONCLUSIONS

eNO was elevated in moderate to severe asthmatics. Withtherapy, eNO started declining in the first week; and by 4weeks, eNO in moderate to severe asthmatics was no differ-ent from that in control subjects. There was decreased IFN-γexpression by the CD4- and CD8-positive PBL of moder-ate to severe asthmatics. Elevated eNO was associated withsuppression of both Th1 and Th2 cytokine expression by thePBL, suggesting a systemic immunomodulatory effect.

ACKNOWLEDGMENTS

The authors sincerely thank Dr. Donald E. Wesson for hiscontinued support for this project. The authors also thank Dr.Ann Hawkins and Dr. Lawrence L. Baldwin for reviewing themanuscript and Cathy Hudson for her assistance with eNOmeasurement.

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