JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 16:357-364 (1996)Mary Ann Liebert, Inc.

Effect of Therapy with Recombinant Human Interferon-y on

the Release of Nitric Oxide by Neutrophils and MononuclearCells from Patients with Chronic Granulomatous Disease

ANTONIO CONDINO-NETO,1 MARCELO N. MUSCARÁ,2 RAQUEL BELLINATI-PIRES,3MAGDA M.S. CARNEIRO-SAMPAIO,4 ANA C. BRANDÄO,5 ANETE S. GRUMACH,5 and

GILBERTO DE NUCCI2

ABSTRACT

The aim of this study was to investigate the effect of recombinant human interferon-y (rHuIFN-y) therapyon the release of nitric oxide (NO) by neutrophils (NEU) and mononuclear cells (MON) from patients withchronic granulomatous disease (CGD). Five patients with this rare disease received rHuIFN-y (50 ¿ig/m2 ofbody surface, given by subcutaneous injection three times a week) for 6 months. Clinical and laboratory eval-uations were performed before and after 1 and 6 months of rHuIFN-y therapy. Nitric oxide release by NEUand MON was assessed by the ability of these cells to inhibit thrombin-induced washed platelet aggregation.The nitrite (N02~) and nitrate (N03~) levels in the supernatant of cultured NEU and MON, as well as inplasma and urine (24 h diuresis), were quantiiled by high-performance liquid chromatography (HPLC).Conventional immunologie tests for assessing phagocyte and lymphocyte functions and humoral immunitywere also performed. Therapy with rHuIFN-y for 6 months did not enhance NO synthesis by NEU or MONfrom the patients with CGD. The urinary but not plasma levels ofNO2 and NO3 were elevated after rHuIFN-y therapy. Phagocyte and lymphocyte functions as well as humoral immunity were not affected by rHuIFN-y therapy. Although few patients were available for the study, we conclude that therapy with rHuIFN-y for6 months did not enhance the synthesis of NO by NEU and MON in CGD patients. Whether the increasedexcretion of N02~ and N03~ in the urine of CGD patients after rHUIFN-y therapy reflects an induction ofNO-synthase in cells other than leukocytes remains to be investigated.

INTRODUCTION ulomatous disease (CGD),(6) a rare and heterogeneous group ofgenetic disorders characterized by recurrent life-threatening

Phagocytes produce two types of inorganic cytotoxic ox- suppurative infections resulting from deficient microbialidants: reactive oxygen intermediates (ROI)<1,2' and reac- killing/7,8' The principal biochemical defect in this disease is

tive nitrogen intermediates (RNI).(3,4) Reactive oxygen inter- an impairment in the production.of ROI by phagocytes16' as a

mediates arise from the univalent reduction of oxygen (O2) to result of genetically induced molecular defects in the NADPH-superoxide anion (02~), hydrogen peroxide (H2O2), and hy- oxidase complex/9'droxyl radical (OH-), catalyzed by the nicotinamide adenine The administration of interferon-y (IFN-y) in vivo or in vitrodinucleotide phosphate (NADPH)-oxidase enzymatic com- enhances both the production of ROI and the killing of bacte-plex.(2' Reactive nitrogen intermediates originate from the NO- rial and protozoan pathogens by phagocytes/10,11' This mech-synthase-catalyzed oxidation of the guanidine nitrogen atom of anism has been the basis for the use of IFN-y in clinical prac-L-arginine, leading to the formation of stoichiometric amounts tice. Therapy with recombinant human (rHu)IFN-ywas recentlyof nitric oxide (NO) and L-citrulline/5' used in CGD patients with promising results because it reduces

The clinical relevance of ROI is illustrated by chronic gran- the relative risk of severe infections by 70% in these patients.

Departments of 'Pediatrics and 2Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, Sâo Paulo, Brazil.3Adolpho Lutz Institute, University of Säo Paulo, Sao Paulo, Brazil.4Department of Immunology, Institute of Biomédical Sciences, University of Sào Paulo, Säo Paulo, Brazil.'Department of Pediatrics, Faculty of Medicine, University of Säo Paulo, Säo Paulo, Brazil.

(12)

357

358

However, the mechanisms by which rHuIFN-y produces a clin-ical improvement in CGD are still unclear/12-16'

Murine macrophages can be activated by a variety of medi-ators to express NO-mediated cytoxicity. In general, this acti-vation requires a priming agent, such as IFN-y/5' followed bya second signal, such as bacterial lipopolysaccharide (LPS), toinduce NO synthesis/17' The reaction between 02~ and NO can

lead to the formation of peroxynitrite (ONOO), a potent cy-totoxic agent/18'

Considering that NEU and MON from CGD patients releaseNO/19' that IFN-y induces RNI synthesis by murinemacrophages in vitro,(5' and that rHuIFN-y therapy reduces therisk of severe infections in CGD patients/12' the aim of thiswork was to investigate the effect of rHuIFN-y therapy on therelease of NO by neutrophils (NEU) and mononuclear cells(MON) from patients with CGD.

MATERIALS AND METHODS

ReagentsThe composition of the Krebs solution was (mM): NaCl 137,

ICC1 2.7, NaHC03 11.9, NaH2P04 0.3, MgS04 0.8, and glu-cose 5.6. The Hank's solution consisted of (mM) NaCl 137,KC1 2.7, Na2HP04 0.5, KH2P04 0.4, and glucose 5.5 and was

supplemented with 10 mM HEPES (HHBSS) adjusted to pH7.4. Superoxide dismutase (SOD, from bovine erythrocytes), L-arginine (free base), W-nitro-L-arginine methyl ester (L-NAME), HEPES, Histopaque 1119, Histopaque 1077, phorbol12-myristate 13-acetate (PMA), zymosan (ZYM), cytochromec (type III), A^-nitro-D-arginine methyl ester (D-NAME),thrombin (bovine), Ham's F10 complete medium, LPS(026:B6), nitroblue tetrazolium (NBT), acridine orange, fluo-rescein antimouse-labeled conjugate, RPMI 1640 medium, andindomethacin were obtained from Sigma (St. Louis, MO).Iloprost was a gift from Schering (Frankfurt, Germany). Mouseanti-CD4 or anti-CD8 monoclonal antibodies were purchasedfrom Dako immunoglobulins A/S (Copenhagen, Denmark).Chromium (51Cr) and tritiated thimidine (3HTd) were purchasedfrom IPEN (Sao Paulo, Brazil). Phytohemagglutinin (PHA) wasobtained from Difco (New York, NY).

Selection ofpatients and healthy controlsFive CGD patients (four males and one female aged 3-23

years, height 90-160 cm, and weight 10-60 kg) were selectedbased upon the following criteria: a clinical history of recurrentsevere suppurative infections, the presence of granulomas as

determined by histopathologic biopsies, and an impaired phago-cyte 02-release stimulated by both soluble and paniculate stim-uli (less than 10% from healthy control values)/20' Other cri-teria for eligibility included preserved renal, liver, andhématologie functions and the signing (by the patients or theirparents) of a form giving informed consent. None of the pa-tients had previously received rHuIFN-y therapy. Patients usedprophylactic trimethroprirn-sulfamethoxazole but no otherforms of immunotherapy or blood derivatives. Patients were hu-man immunodeficiency virus negative and were in a clinicallysuitable condition. Healthy male controls ranging in age from22 to 28 years were selected after clinical and laboratory screen-

CONDINO-NETO ET AL.

ing. The study was approved by the medical school ethics com-

mittee.

Study designRecombinant human interferon-y (Boehringer Ingelheim,

Germany; specific activity of 3 X 107 U per milligram proteinat a concentration of 0.2 mg/ml) was provided as a sterile so-

lution ready for injection. The patients received subcutaneousinjections of rHuIFN-y (50 pg/m2 of body surface) three timesa week and were submitted to a clinical and laboratory follow-up before and after 1 and 6 months of rHuIFN-y therapy.

The experimental laboratory assays included determinationof (1) the release of NO by NEU and MON as assessed by theability of these cells to inhibit thrombin-induced washed plateletaggregation, (2) the release of NÛ2- and N03~ by culturedNEU and MON, (3) the levels of N02 and N03~ in the plasmaand urine (24 h diuresis), (4) the release of 02~ by NEU andMON, (5) neutrophil functional assays (chemotaxis, phagocy-tosis, bactericidal activity, and NBT test), (6) lymphocyte countand functional assays (CD4/CD8 ratio, blastogenic activity, andnatural killer cell activity), and (7) humoral immunity (serumimmunoglobulins, IgG subclasses, and complement levels).

Preparation of washed plateletsBlood from healthy controls was collected into plastic flasks

containing sodium citrate (3.8%). Platelets were washed in oxy-genated (95% 02 and 5% C02) Krebs solution containing ilo-prost (300 ng/ml) as described by Radomski and Moneada/21'The platelet suspension was adjusted to 1 X 108 cells/ml inKrebs solution containing indomethacin (10 pM, to inhibit theformation of cyclooxygenase products) and CaCl2 ( 1 mM).

Isolation of neutrophils and mononuclear cells

Neutrophils and MON from healthy controls and from CGDpatients were obtained as pure cell populations using a SigmaHistopaque discontinuous density gradient kit (density 1.077and 1.119 g/ml) according to the manufacturer's instructions/22'The leukocyte suspensions were adjusted to give the desiredconcentration of cells for each experiment in HHBSS (pH 7.4)containing 10 pM indomethacin and 1 mM CaCl2.

Nitric oxide release by neutrophils and mononuclearcells

The release of NO by NEU and MON from CGD patientsand from healthy controls was assessed by the ability of theseleukocytes to inhibit thrombin-induced washed platelet aggre-gation/23' Briefly, a suspension of 1 X 108 washed plateletsml-1 (500 p\) was incubated at 37°C for 1 minute in a Paytondual-channel aggregometer with continuous stirring at 900 rpmand then stimulated with thrombin (50-200 mU/ml). The de-crease in optical density was recorded for 5 minutes after theaddition of thrombin.

Neutrophils orMON (5 X 107 cells/ml) in a volume of 10-50pi were added to the platelet suspension and the incubation con-

tinued for a further 1 minute before stimulation with thrombin.The final leukocyte concentration ranged from 1 to 5 X 106cells/ml. Superoxide dismutase (60 U/ml) was added immedi-ately before the leukocytes. Neutrophils and MON (5 X 107

EFFECT OF rHuIFN-y THERAPY ON NITRIC OXIDE RELEASE BY CGD LEUKOCYTES

cells/ml) were preincubated (60 minutes, room temperature)with L-arginine (1 mM), the NO synthesis inhibitor L-NAME(300 ¿iM)/24' its enantiomer (D-NAME, 300 pM), or with bothL-arginine (1 mM) and L-NAME (300 pM) before the cellswere added to the platelet suspension.

Nitrite and nitrate levels in the supernatant of culturedneutrophils and mononuclear cells and in plasma andurine

Neutrophils and MON (1.4 X 106 ml"') from CGD patientswere cultured (duplicate) in Ham's F10 complete medium for24 h (37°C, humid atmosphere containing 5% CO2) in the pres-ence of (1) patient's serum (10% vol/vol), (2) patient]s serum

and LPS (100 jug/ml), (3) patient's serum, LPS, and L-NAME(300 pM), or (4) patient's serum, LPS, and D-NAME (300pM). Ham's F10 complete medium contained L-arginine (1.2mM). Neutrophils and MON from healthy controls were cul-tured under the same experimental conditions (serum was ob-tained from healthy controls).

Nitrite and NO3- concentrations in the supernatants of cul-tured NEU and MON and in plasma and urine (24 h diuresis)were simultaneously determined using an on-line high-perfor-mance liquid chromatography (HPLC) system with two post-column reactions, which allowed the separation of N03~ andN02~ and their specific quantification/25' The postcolumn re-

actions involved (1) the reduction of N03~ to N02~ on a cop-per-plated cadmium powder-filled column and (2) the forma-tion of the diazo compound after reaction with the Griessreagent. All these steps were carried out on-line, and peak ab-sorbances were monitored at 540 nm.

In all cases, the peaks for NÛ2~ and N03~ were satisfacto-rily separated by the mobile phase used. The retention times forN02" and N03- were, 8.4 ± 0.2 and 10.3 ± 0.3 minutes, re-spectively. The sensitivity of the method was 80 nM (equiva-lent to 32 pmol N02~ or N03~), based on a signal-to-noise ra-

tio of 5, and as little as 0.05-0.1 ml sample volume was

required.

Superoxide anión release by neutrophils andmononuclear cells

The release of 02" by NEU and MON (from CGD patientsand from healthy controls) was assessed in duplicate by a SOD(90 U/ml) inhibitable cytochrome c (80 /xM) reduction assay/26'The neutrophil or mononuclear cell suspension (1.4 X 106mr1) was stimulated with either PMA (30 nM) or ZYM (100particles/cell) for 60 minutes at 37°C. The absorbance of thesupernatants was monitored at 550 nm. The amount of 02~ re-

leased was calculated using an extinction coefficient of 21,100M-1 cm-1. The results were expressed as nmol 02~ 1.4 X 106cells/h.

Neutrophil chemotaxis, phagocytosis, bactericidalactivity, and NBT assays

Neutrophil (2 X 106 cells/ml) chemotactic assays were per-formed in duplicate by the method of Boyden/27' The chemo-tactic factor was individually obtained by the activation of freshCGD patient serum (10% vol/vol in HHBSS) or fresh healthycontrol serum (10% vol/vol in HHBSS) with LPS (100 /ug/ml)

for 30 minutes at 37°C. The cells were then incubated with therespective chemotactic factor for 2 h in a humid atmospherecontaining 5% CO2 at 37°C. The results were expressed as thedistance (/am) between the upper cell layer and the lower fieldoccupied by at least three cells (average of 20 measurements).

Neutrophil phagocytosis and bactericidal activity assayswere assessed in triplicate by a fluorochrome assay/28' Equalvolumes of Staphylococcus aureus Cowan I suspensions (2 X107 bacteria/ml) opsonized with CGD patient or healthy con-

trol serum were incubated with the respective CGD or healthycontrol neutrophil suspension (2 X 106 cells/ml) in a rotatorywater bath shaker at 150 rpm for 15 minutes at 37°C. The cellswere then stained with acridine orange (14.4 mg/liter), washed,and examined. The number of cells phagocytozing at least threebacteria (phagocytic activity) and the number of viable (greenfluorescence) and nonviable (red/yellow fluorescence) intracel-lular bacteria were simultaneously recorded (phagocytic ca-

pacity). The results were expressed according to the followingformulas: phagocytic index = (phagocytic activity) X (phago-cytic capacity) X 100-1; percentage of killed bacteria = (num-ber of dead bacteria) X (number of dead + live bacteria)-1 X100.

Neutrophil NBT tests were performed by incubating CGDpatient or healthy control neutrophil suspensions (2 X 106cells/ml) with PMA (30 nM) for 15 minutes at 37°C and thenincubating this suspension with NBT (300 pglml) for 15 min-utes at 37CC/29' The results were expressed as a percentage ofcells that exhibited NBT-reduced crystals in the cytoplasm.

Lymphocyte CDyCDg ratio, blastogenic activity, andnatural killer activity

Lymphocyte counts (CD4 and CD8) were assessed by ini-tially incubating MON from CGD patients (2 X 106 cells/ml)with mouse anti-CD4 or anti-CD8 monoclonal antibodies (1 hat 37°C; 5% C02), followed by a further incubation with a flu-orescent antimouse-labeled conjugate (1 h at 37°C; 5% CO2)as previously described/30'

Lymphocyte proliferative activity (from CGD patients andfrom healthy controls) was assessed by a standard 3HTd uptakeassay under PHA stimulation/3" The results were expressed as

the stimulation index = (counts per minute in the presence ofPHA) X (counts per minute in the absence of PHA)-1.

Natural killer cell activity (from CGD patients and fromhealthy controls) was assessed using a standard 51Cr release as-

say (50:1, 25:1, and 12:1 ratios)/32' The results were expressedas percentage cytotoxicity = (test counts per minute

—

sponta-neous counts per minute) X (total counts per minute

—

sponta-neous counts per minute)^1 X 100.

Serum immunoglobulin and IgG subclass levels andcomplement hemolytic activity

Serum immunoglobulin and IgG subclass levels from CGDpatients were determined by radial immunodiffusion'33' usingBoehringer Nor-Partigen plates for IgA, IgM, and IgG(Boehringer-Ingelheim, Germany) and the monoclonal anti-bodies JK512, GOM1, ZG4, and RJ4 (Oxoid, England) forIgGi, IgG2, IgG3, and IgG4 determinations, respectively. Theresults were compared with those of healthy Brazilian chil-dren/34' Complement hemolytic titration of the classic pathway

360 CONDINO-NETO ET AL.

Table 1. Spontaneous and PMA- (30 nM) and Zymosan(100 Particles/Cell)-Stimulated Release of SuperoxideAnión (nmol per 1.4 X 106 Cells per h) by Neutrophilsand Mononuclear Cells from Patients with Chronic

Granulomatous Disease Before and After 6 Months of

Recombinant Human Interferon-y Therapy"

No drug 6 Months rHu/FN-y

Patients SPONT PMA ZYM SPONT PMA ZYM

NeutrophilsRCSLFRGBTPJEMACSA

000.100.1

Mononuclear cellsRCSLFGRBTPJEMACSA

1.40.600.30.2

•0.20.80.20.30.2

0.25.50.200.8

00.60.10.40.1

2.13.10.10.80.5

00.100.1ND

0.20.60.10ND

0.21.20.30.5ND

1.34.80.90.8ND

0.10.80.10.3ND

0.72.60.40.4ND

aND, no data.

(CH5o) from CGD patients was determined according to themethod of Mayer/35'Statistics

Results are represented as the minimum, median, and max-

imum. Comparisons among groups were performed by theKruskal-Wallis test/36' Values ofp < 0.05 were considered sig-nificant.

RESULTS

During the study period one patient died as a result of res-piratory failure (month 3), whereas the others remained in a sta-ble clinical condition and had no severe infections. All patientsdemonstrated the following side effects toward rHuIFN-y mod-erate fever, shivers, nausea, lack of appetite, headache, andmyalgia. These symptoms appeared 2 h after the injection ofrHuIFN-y and were alleviated by the administration of parac-etamol. No other side effects were reported throughout thestudy, and the rHuIFN-y was well tolerated by all individuals.The results of the routine clinical tests stipulated for assessingdrug toxicity in this study (transaminases, urea and creatininelevels, hématologie counts, and urinalysis) were all within thenormal ranges before and 6 months after treatment withrHuIFN-y.

Neutrophils and MON from CGD patients had a low spon-taneous and zymosan- or PMA-stimulated O2" release through-out the study (Table 1). The values for the neutrophil NBT testswere below 5% after rHuIFN-y therapy.

Nitric oxide release by neutrophils and mononuclearcells bioassay

The minimum concentration of thrombin required to induceirreversible washed platelet aggregation within 5 minutes un-

der our experimental conditions varied from 50 to 200 mU/ml(Figs. 1 and 2). Incubation of platelets (1 X 108 ml-1) withNEU or MON from CGD patients (1-5 X 106 cells/ml) for 1minute before stimulation with thrombin resulted in an inhibi-tion of platelet aggregation that was proportional to the num-

ber of leukocytes added (results not shown). This inhibition was

potentiated by SOD (60 U/ml, results not shown). Preincubation

NO DRUG(3.2 x106 cells ml"1)

ONE MONTH rhIFN-Y(4.8 x106 cells ml-1)

SIX MONTHS rhIFN-Y(5.0 x10s cells ml"1)

L-ARGL-NAME + L-ARGD-NAME

L-NAME

T 50 mU/ml

L-ARGL-NAME + L-ARGD-NAME

L-NAME

T50mU/ml

L-ARG

T 50 mU/ml

FIG. 1. Neutrophils from patients with chronic granulomatous disease (representative trace of patient RCS) inhibited throm-bin-induced (50 mU/ml) washed platelet aggregation before (3.2 X 106 cells/ml) and after 1 month (4.8 X 106 cells/ml), but notafter 6 months (5.0 X 106 cells/ml) of recombinant human interferon-y therapy. /v,<"-nitro-L-arginine methyl ester (L-NAME, 300pM), but not N^-nitro-D-arginine methyl ester (D-NAME, 300 pM), reduced the inhibitory effect of the neutrophils on plateletaggregation (1 X 108 ml-1). This reduction was reversed by simultaneously preincubating the neutrophils with L-NAME (300pM) and L-arginine (1 mM). L-arginine alone (1 mM) did not potentiate the basal release of nitric oxide by neutrophils fromchronic granulomatous disease patients throughout the study.

EFFECT OF rHuIFN-y THERAPY ON NITRIC OXIDE RELEASE BY CGD LEUKOCYTES 361

NO DRUG(2.8 x106 cells/ml)

ONE MONTH rhlFN-y(4.0 x106 cells/ml)

SIX MONTHS rhIFN-Y(4.0 x106 cells/ml)

L-ARGL-NAME + L-ARGD-NAME

L-NAME

T 50 mU/ml

L-ARGT 50 mU/ml

L-ARGT 50 mU/ml

FIG. 2. Mononuclear cells from patients with chronic granulomatous disease (representative trace of patient RCS) inhibitedthrombin-induced (50 mU/ml) washed platelet aggregation before (2.8 X 106 cells/ml), but not after 1 month (4.0 X 106 cells/ml)or 6 months (4.0 X 106 cells/ml) of recombinant human interferon-y therapy. Ar-nitro-L-arginine methyl ester (L-NAME, 300pM), but not iV-nitro-D-arginine methyl ester (D-NAME, 300 pM), reduced the inhibitory effect of mononuclear cells on washedhuman platelet aggregation (1 X 108 ml-1). This reduction was reversed by simultaneously incubating the mononuclear cells withL-NAME (300 pM) and L-arginine (1 mM). L-Arginine alone (1 mM) did not potentiate the basal release of nitric oxide bymononuclear cells from chronic granulomatous disease patients throughout the study.

(60 minutes at room temperature) of NEU and MON from CGDpatients with the nitric oxide synthesis inhibitor L-NAME (300pM) but not with D-NAME (300 pM) reduced the capacity ofthese cells to inhibit platelet aggregation. This reduction was

reversed by simultaneously preincubating NEU and MON withL-NAME (300 pM) and L-arginine (1 mM, Figs. 1 and 2).Preincubation of NEU and MON from CGD patients with L-arginine alone (1 mM) did not potentiate the inhibitory effectof these leukocytes on platelet aggregation. We were unable todetect the release of NO by MON following 1 month of rHuIFN-y therapy or by either NEU or MON after 6 months of rHuIFN-y therapy (Figs. 1 and 2).

Nitrite and nitrate levels in the supernatant of culturedneutrophils and mononuclear cells and in plasma andurine

The levels of NO2- and N03~ in the supernatants of cul-tured NEU and MON from patients with CGD remained lowthroughout the study. The maximum amounts of NÛ2~ andN03- released by NEU from patients with CGD before and af-ter 1 and 6 months of rHuIFN-y therapy were, respectively(nmol N02~/ N03- per 7 X 105 cells/ml/24 h, in the presenceof patient's serum and LPS, 2.46/1.20,1.86/1.34, and 2.25/1.23.The maximum amounts of N02~ and N03~ released by MONfrom patients with CGD before and after 1 and 6 months ofrHuIFN-y therapy were, respectively (nmol NÛ2-/ N03- per7 X 105 cells/ml/24 h, in the presence of patient's serum andLPS), 1.52/1.49, 0.13/1.37, and 0.85/1.93. The maximumamounts of NÛ2~ and N03~ released by healthy control NEU

and MON were, respectively (nmol N02 / N03 per 7 X 105cells/ml/24 h, in the presence of healthy control serum and LPS,n = 10), 4.37/3.48 and 2.79/2.89.

After 1 month of rHuIFN-y therapy, two patients presented,respectively, 0.26 and 0.32 pmo\ N02~ per 24 h in the urine(N02~ was not detected in the urine of CGD patients beforerHuIFN-y therapy). After 6 months, all four patients presenteddetectable levels of NÛ2~ in the urine (median value was 0.30pmo\ N02~ per 24 h). There was also an increase in the lev-els of N03~ in the urine of these patients after rHuIFN-y ther-apy (median values were, respectively, 423.5, 522.0, and 648.0pmo\ N03- per 24 h), although it was not statistically signif-icant (p > 0.05; Kruskal-Wallis test, calculated H = 3.380 <critical H = 5.666 forp = 0.05). All patients had negative urinecultures.

The plasma N02~ and N03~ levels (pM) of CGD patients(respective median values before and after 1 and 6 months ofrHuIFN-y therapy: 0.33/15.70, 0.06/14.57, and 0.90/14.25)were not affected throughout the study (p > 0.05, Kruskal-Wallis test, calculated H for N02~ and N03~ values were, re-spectively, 4.326 and 0.223 < critical H = 5.666 forp = 0.05).

Neutrophil chemotaxis, phagocytosis, and bactericidalactivity

The spontaneous migration and chemotaxis (pm) of neu-

trophils from CGD patients (respective median values before andafter 1 and 6 months of rHuINF-y therapy: 34/63, 41/73, and 37/80)were not affected throughout the study (p > 0.05, Kruskal-Wallistest, calculated H for spontaneous migration and for chemotaxis

362 CONDINO-NETO ET AL.

were, respectively, 3.311 and 1.000 < critical H = 5.666 for/? =

0.05) and were significantly lower than those observed in NEUfrom healthy controls (p < 0.05, Kruskal-Wallis test, calculatedH for spontaneous migration and for chemotaxis were, respec-tively, 8.175 and 15.319 > critical H = 7.814 forp = 0.05, me-

dian healthy control neutrophil spontaneous migration andchemotaxis values were, respectively, 45 and 100, n

—

14).The phagocytic index and bactericidal activity (% killed bac-

teria) of neutrophils from CGD patients (respective median val-ues before and after 1 and 6 months of rHuIFN-y therapy:10.3/34.4, 12.3/38.2, and 16.0/36.7) were not affected through-out the study (p > 0.05", Kruskal-Wallis test, calculated H forphagocytic index and for bactericidal activity were, respec-tively, 1.620 and 0.385 < critical H = 5.666 forp = 0.05). Thephagocytic index of neutrophils from CGD patients was sig-nificantly higher than that observed in NEU from healthy con-

trols (p < 0.05, Kruskal-Wallis test, calculated H = 9.060 >critical H = 7.814 forp = 0.05, median value for healthy con-

trol neutrophil phagocytic index was 6.7, n = 14). The bacte-ricidal activity of neutrophils from CGD patients was signifi-cantly lower than that observed in NEU from healthy controls(p < 0.05, Kruskal-Wallis test, calculated H = 14.824 > criti-cal H = 7.814 for p = 0.05, median value for healthy controlbactericidal activity was 54.3, n = 14).

Lymphocyte CD^/CDg ratio, blastogenic activity, andnatural killer activity

Lymphocyte CDVCDs ratios from CGD patients (respectivemedian values before and after 1 and 6 months of rHuIFN-y.1.4, 1.6, and 1.6) were within the normal range'37' and were notaffected throughout the study (p > 0.05, Kruskal-Wallis test,calculated H = 4.063 < critical H = 5.666 for p = 0.05).

The lymphocyte proliferative activity (stimulation index)from CGD patients (respective median values before and after1 and 6 months of rHuIFN-y therapy: 128.4, 87.5, and 98.4)was not affected throughout the study (p > 0.05, Kruskal-Wallis test, calculated H 0.223 < critical H = 5.666 for p =

0.05) and was similar to that observed in lymphocytes fromhealthy controls (p > 0.05, Kruskal-Wallis test, calculated H =

2.631 < critical H = 7.814 for p = 0.05, median value forhealthy control proliferative activity was 63.7, n = 14).

The natural killer cell activity (% cytotoxicity: 50:1, 25:1,and 12:1 ratios) of CGD patients (respective median values be-fore and after 1 and 6 months of rHuIFN-y therapy:31.2/21.6/10.2, 52.9/39.4/20.1, and 55/43.1/28.1) was not af-fected throughout the study (p > 0.05, Kruskal-Wallis test, cal-culated H for 50:1, 25:1, and 12:1 ratios were, respectively,2.965, 3.651, and 5.400 < critical H = 5.666 forp = 0.05) andwas similar to that observed in lymphocytes from healthy con-

trols (p > 0.05, Kruskal-Wallis test, calculated H for 50:1,25:1,and 12:1 ratios were, respectively, 3.003, 3.838, and 5.407 <critical H = 7.814 forp = 0.05, median values for healthy con-

trol natural killer cell activity in 50:1, 25:1, and 12:1 ratios were,respectively, 46.2, 33.7, and 14.5, n = 14).

Serum immunoglobulin and IgG subclass levels andcomplement hemolytic activity

The serum immunoglobulin levels (mg/dl) of CGD patients(respective median values for IgA, IgG, and IgM before and af-

ter 1 and 6 months of rHuIFN-y therapy: 680/2450/200,552/2330/136, and 697/1725/132) were not affected through-out the study (p > 0.05, Kruskal-Wallis test, calculated H forIgA, IgG, and IgM were, respectively, 0.081, 1.179, and0.325 < critical H = 5.666 for p = 0.05). Similarly, IgG sub-classes (mg/dl, respective median values for IgGi, IgG2, IgG3,and IgG4 before and after 1 and 6 months of rHuIFN-y2300/432/86/11, 2300/400/52/12, and 1225/398/49/11) were

also not affected throughout the study (p > 0.05, Kruskal-Wallis test, calculated H for IgGi, IgG2, IgG3, and IgG4 was,respectively, 5.579, 1.348, 1.090, and 0.248 < critical H =

5.666 for p = 0.05). All CGD patients presented hyperglobu-linemia compared with healthy Brazilian children/34'

The complement hemolytic activity (CH50, U/ml) of CGDpatients (respective median values before and after 1 and 6months of rHuIFN-y, 107, 103, and 114) were not affectedthroughout the study (p > 0.05, Kruskal-Wallis test, calculatedH = 0.725 < critical H = 5.666 forp = 0.05) and were withinthe normal range/38'

DISCUSSION

Our results clearly demonstrate that the CGD patients ex-

hibited a very low neutrophil and mononuclear cell 02~ pro-duction, neutrophil NBT tests below 5%, an impaired neutrophil6actericidal activity, a lower neutrophil chemotactic activity, a

higher neutrophil phagocytic index, normal lymphocyte func-tions as assessed by the CD^CDg ratio, proliferative activity,and natural killer cell activity, hypergammaglobulinemia, andnormal complement hemolytic activity compared with healthycontrols. These parameters were not affected by rHuIFN-y ther-apy, which is in accordance with the literature/12-15,39,40'

Most of these CGD patients demonstrated mild symptoms,such as moderate fever, shivers, nausea, lack of appetite,headache, and myalgia, after rHuIFN-y injections, all of whichwere alleviated by administration of the cyclooxygenase in-hibitor paracetamol/12' Cytokines induce the expression of cy-clooxygenase type 2 (COX-2)/41' and paracetamol blocksCOX-2 activity/42,43' Because IFN-y induces the release ofeicosanoids by healthy control or CGD leukocytes in vitro/44^the moderate fever observed raises the possibility that therHuIFN-y therapy induced COX-2 in vivo.

Before rHuIFN-y therapy, NEU and MON from CGD pa-tients inhibited thrombin-induced washed platelet aggregation.This inhibition was caused by the release of NO because it wasinhibited by L-NAME but not by D-NAME and could be re-

versed by L-arginine and potentiated by SOD/19,23,24' Despitethe considerable amount of in vitro evidence demonstrating theability of IFN-y to induce the biosynthesis of large amounts ofNO in murine macrophages and the importance of NO in mi-crobicidal mechanisms/5,45,46' our results demonstrate thattreating CGD patients with rHuIFN-y for 6 months did not po-tentiate the release of NO by their NEU and MON as deter-mined by the ability of these cells to inhibit thrombin-inducedwashed platelet aggregation and also by the release of NObreakdown products (the levels of NÛ2~ and N03 released bycultured NEU and MON from CGD patients were close to thelower detection threshold of the method and were very similarto those released by healthy control leukocytes). Furthermore,

EFFECT OF rHuIFN-y THERAPY ON NITRIC OXIDE RELEASE BY CGD LEUKOCYTES 363

we were unable to detect the release of NO by MON from thesepatients following 1 month of rHuIFN-y therapy and by bothMON and NEU after 6 months of rHuIFN-y therapy.Cyclooxygenase products did not affect our results because in-domethacin (10 pM) was present in all experiments. It is un-

likely that the failure to detect NO release by NEU and MONfrom CGD patients was caused by technical problems becausein all experiments, the NO-releasing ability of NEU and MONfrom healthy controls was assayed as a positive control. The fail-ure to detect NO release by NEU and MON from CGD patientsfollowing rHuIFN-y therapy is currently under investigation.

Murray and Teitelbauir/47' and Schneemann et al/48' madethe interesting observation that rHuIFN-y was not able to inducethe synthesis of RNI by human MON in vitro and that such cellsfrom patients with acquired immunodeficiency syndrome whohad been treated with rHuIFN-y for 3 weeks also did not releasea great amount of RNI. These authors concluded that NO was

not important as a defense mechanism in humans. These obser-vations are contrary to those made in vitro by Malawista et al./49'who demonstrated the importance of RNI as a bactericidal mech-anism against Staphylococcus aureus in human neutrophil cyto-plasts, including those derived from CGD patients.

We have observed an increase in the urinary levels of NO2-and N03- in our patients after rHuIFN-y therapy. Human chon-drocytes'50' and hepatocytes*51' synthesize large amounts of RNIwhen stimulated by rHuIFN-y and LPS. These observations raisethe interesting possibility that treating humans with rHuIFN-ymay induce the biosynthesis of significant amounts of NO in vivoin these and other cells (here possibly illustrated by cells from therenal-genitourinary tract), thereby providing a nonspecific tissuedefense mechanism independent of the presence of cells from thehost's immunologie system. It therefore seems profitable, in a fu-ture study, to examine the expression of NO-synthase and otherprotective factors at locations other than the cells of the immunesystem in CGD patients treated with rHuIFN-y.

Although few patients have been treated, we conclude thattherapy with rHuIFN-y does not stimulate the leukocytes fromCGD patients to release NO, nor does it have any influence on

classic immunologie functions. Thus, the mechanism by whichrHuIFN-y benefits CGD patients remains unclear. Despite the invitro evidence that RNI play an important role in phagocytic cy-totoxicity, we believe that it would be much easier to define sucha relationship if one were able to identify a human immunodefi-ciency condition characterized by a lack of RNI production.

ACKNOWLEDGMENTS

We thank Boehringer de Angeli Ltda. for kindly providingthe rHuIFN-y, Dr. Constancia L. Diogo for her technical as-

sistance, and Dr. Alberto J. S. Duarte for allowing us to use hislaboratory for the cellular immunology tests. We also thank Dr.Stephen Hyslop for the linguistic review of the manuscript.

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Received 21 July 1995/Accepted 3 January 1996