Clin. exp, Immunol. (1992) 88, 203-206

Treatment of patients with chronic granulomatous disease with reeombinant human interferon-gamma does not improve neutrophil oxidative metabolism, cytochrome b558 content or levels of four anti-microbial proteins T. J. MUHLEBACH*, J. GABAYt, C.F. NATHANt, C, ERNY*. G, DOPFERJ. H. SCHROTEN§. V. WAHN§ & R. A. SEGER* "Division of Immunology Haetnatotngy, University Chitdreti's Hospitul, Ziirich, Switzertand. ^Division of Hematotogy-Oncology, Department of Medicine, Cortwll University Medicat College. New York. NY. USA, XUniv. Kinderklinik. Tiibingen. and^Vniv. Kinderklinik. DUssetdorf. Germany

i Accepted for publication 15 January 1992)

SUMMARY Recombinani interferon-gamma (rIFN-y) has been described lo enhance phagocyte functions in vitro and in rivo in several patients with chrotiic granulomatous disease (CGD). To demonstrate the clinical usefulness of this treatment. 128 patients were treated in a randomized, double-blind multicentre study with a placebo preparation or with rIFN-)'. We analysed parameters of neutrophil oxidative and non-oxidative metabolism in 16 patients enrolled in this study. No enhanced superoxide-release was observed in patients treated wilh rlFN--; compared to placebo-treated patients. Phagocyte cytochrome bfl58 content also remained unchanged. Levels of four nonoxidative antimicrobial proteins (cathepsine G. azurocidine. p29b, lactolerrin) rose, tell, or remained unchanged, irrespective of treatment with rIFN-y or placebo. Keywords chronic granulomatous disease cytochrome b558 anti-microbial proteins

reeombinant interferon-gamma

placebo-controlled international phase \U study of rlFN-y in patients with CGD was performed showing clinical usefulness of the drug for infection prophylaxis [10]. Here we demonstrate analyses of NADPH oxidase activity, cytochrome b588 content, anti-microbial protein (AP) levels [II] in neutrophils and of serum IgG subclasses in 16 patienls enrolled in this rlFN-y study.

INTRODUCTION Chronic granulomatous disease (CGD) is a group of rare congenital phagocyte disorders characterized by recurrent pyogenic infections ofpatients with catalase-positive bacteriae ;md fungi. Due to defects in the NADPH oxidase the phagocytic cells of these patients are unable to generate superoxide (O;), hydrogen peroxide and other microbicidal oxygen metabolites in response to ingested microorganisms [1,2]. Reeombinant interferon-gamma (rlFN-v) is a lymphokine that has been shown to activate macrophages. In vitro and in vivo treatment of normal monocytes und neutrophits enhances the production of reactive oxygen intermediates and improves the capacity of killing of phagocytosed microorganisms [3,4]. Recent pilot studies demonstrated a partial correction ofthe phagocyte defect in patient.s with X-Iinked as well as autosotnal recessive CGD by subcutaneous rIFN-y [5-9J. Treatment resulted in tive lo 10-fotd increase in superoxide production of neutrophils and monocytes; bactericidal activity rose proportionally and ihe phagocyle cytochrome b558 contents increased up 10 50"'. ol normal values [6,7]. Subsequently a randomized.

PATIENTS AND METHODS Patients Twelve in feet ion-free patients with eomplete X-linked eytoehrome b558 deficiency (eight unrelated male patients, two cousins and one uncle and his nephew), two patienls, one male, one female, with autosomal-recessive cytochrome B558 deficiency and two patients, one female, one male wilh auiosonialrecessive cytochrome b558-positive CGD were enrolled in the study with local Ethical Committee approval. All 16 patients were on prophylactic antibiotics (co-tritnoxazole) trealtnenl, none received anii-tnycolics or corlicosteroids. Neutrophils from a healthy control were included in each analysis, The diagnosis of CGD has been established by defeclive nitroblue tetrazolium (NBT) reduction in the NBT slide-test afier stimulation with phorbol myristale acetate (PMA) and

Correspondence: R. A, Segcr. Abteilung Immunologie-Haematolojzic, Kindcrspital, Unversitat Zurich, Sleinwiesstr. 75, 8032 Zurich, Switzerland.

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opsonized zymosan particles (OPZ). deficienl oxygen consumption or superoxide release frotn neutrophils upon maximal stimulation with PMA and fMLP. and in most cases by determination of cytocliromc b558 content of neutropliils. Xlinked inheritance has been established by the tnosiac in the NBTtest ofthe mothers of these patienis. A ulosomal-recessive inheritance was diagnosed by normal NBT lest in lhe parents and delermination of cytochrome b5!>8 content and where necessary of soluble cyloso!ic faclors of neuirophils. Study design The complete protocol ofthe study is described elsewhere [10]. Briefly, ihe influence of rlFN-y was investigated in a multicentre, randomized, double-blind, placebo-controlled trial with two parallel groups (designated A and B). The patients received rlFN-7 (50 (igjmr) (group A) or placebo (group B) subcutaneously three limes a week for an average of 9 months in addition to co-lrimoxazole prophylaxis. Neutrophil functions were assayed on day 0.. 90 and 180, Subsequently an interim analysis was performed, and lhe sludy was unblindcd and stopped, because clinical data were clearly in favour o'^ rIFN-y treatments. There has been no violation of the treatment protoeol in our palient group. Nine out of the 16 patients had received the placebo preparation and seven patients received rIFN-;. In lhe placebo group two episodes of serious infection necessitating hospilalization had occurred (one lymphadenitis, one aspergillus pneumonia), and in the IFN group one episode (a case of pneumonia). Laboratory evaluations Neuirophils were isolated from citrated blood sampled before the nexl injection of the study drug [12], NBT reduction was performed as described by BoMcr etai [1] after stimulation with PMA (200 ng/ml) as well as wilh OPZ (150 ;ig/ml). The percentage of forniazan-positive cells was determined by two persons counting three hundred on each slide and averaging the results. Superoxide formation was measured al 37' C as the superoxide dismutase (SOD)-sensitive redution of cytochrome c with PMA (100 figim\) and PMA + I^MLP (I /(M) as stimuli. Cytochrome b558 was measured by dctcrminaiion of the reduced minus oxidized spectrum in a Triton X-100 extract. For all calculations of cytochrome b558 content an extinction coefficienl of 106 mM'cm' for the Sorel band was used [13]. Western blot analysis of cytochrome b was performed according to Vcrhocven et al. [14]. Antibodies used were MoAb 48 against the heavy chain and MoAb 449 against the light chain of cytochrome b558 (Dirk Roos. Amsterdam, The Netherlands): alternatively rabbit aniisera were used from C. Parkos, La Jolla, CA. against lhe light chain and from S. Orkin. Boston. MA, against the heavy chain of cylochrome b.'i58. The Killing Assay wilh Staphylococcus aureus 5O2A was performed according lo the standardized protocol [10], AP levels were determined as follows: neulrophil pellets from heallhy donors or patients at day 0 and day 90 of treatment were resuspended in Krebs Ringer phosphate buffer with glucose in lhe presence of aprotinin (5 /ig/ml), pcpstatin {5 /ig/ml). and PMSF (5 mM) and extracted with 50 niM Tris, pH 6 8, 1 "A. SDS, 5% glycerol. 2% jimercapioethanol at 10' cells/ml. Samples were healed at 100 C for 3 min, centrifuged al 10 000 j? for 10 tnin. and I0-/J1 aliquols (equivalent to 10^ neutrophils) were used for SDS PAGF and Western biol analysis using monospecilic. polyclonal rabbit

antibodies as described [15], Levels of cathepsin G, azurocidine. p29b (all from azurophilic granules) and lactofcrrin (from specific granules) were determined by densitometry as related to standard curves run with O i - I fig of each of Ihc purified proteins. Total IgG was determined ncphclomclrically and IgG subclasses were determined by conventional radial immunodiffusion using subclass-specific polyclonal antisera from Janssen Biochimica. RESULTS Six ofthe X-Iinked cytochrome b558-deficienl patients and one patient with autosoma! 67-kD protein deficiency were treated with rIFN-y. The other six X-linked patients received placebo. The two patients with aulosomal cytochrome b558 deficiency and one patient with 47-kD protein deficiency also received placebo. The results are summarized in Figs 1, 2 and 3. Briefiy, none of the parameters observed changed in the rlFN-y-trealed palienls compared with the placcbo-lreated controls. In lhe eytochemieal NBT test (Fig la) all the cells remained formazan negative in all palienls after stimulation with PMA (nol shown) as well as with OPZ. The deficient superoxide production (Fig, I b) was not improved in patients under rIFN-y treatment. The cytochrome b558 content (Fig. Ic) remained below detection limit in all patients wilh cylochrome b558 deficiency, and was equally unchanged in one rIFN-y-lreated patient with 67-kD protein deficiency. In Western blots prepared from Triton X-100 cell extracts from day 0 and day 180 no change in cither of the two subunits of cylochrome b558 was observed (nol shown). The killing of S. aureus 502A (Fig. Id) was nol significantly increased in rlFN-y-treated patients after 1 h (nol shown) and 2 h of incubation. The total IgG and the IgG subclass levels remained unaffected by rIFN-y treatment (Fig. 2). A marked increase or decrease in the level of one or more ofthe AP occurred over the 90-day interval in 10 out ofthe 16 patients. These changes did not appear to be correlated with treatment status {Fig. 3).

DISCUSSION In contrast to earlier reporls [5-9] we could nol observe any improvement of neutrophil microbicidal functions under therapy with rIFN-v in six CGD patients with X-llnkcd cytochrome b558 deficiency (the most cotiimon genetic form of the disease) and one paticnl with 67-kD protein deficiency. The palicnts described in the earlier reporls could have been special responders, e.g, variants of CGD wilh a defect that might be partially corrected by increased expression of lhe relevant proteins. Increased produclon of mRNA ofa defective componenl may in most cases however not lead to expression of a functional protein. The itnproved clinical state of patients treated wilh rIFN-y is ihus unexplained, but might be related to the ability of the cylokinc to induce increased levels of respiratory burst independent antimicrobial activity, either under basal conditions, or after additional stimuli generated during lhe course of infection. Altempls lo measure AP during infection might be complicated by partial activation and degranulation of ncutrophits in ihc circulation. Indeed, responses lo inapparent infeclion may have contributed to the fluctuations observed in the levels of AP in mosl CGD patients over a period of 90 days. The pattern of

Treatment of CGD patients with rIFN-y

205

120 100 80 60 40 20 0

Control

Xb-/P Xb-/lFN Ab-/P 67 kCi/IFN

Conlrol

Xb-/P ln-61

Xb-/1FN (n-6)

Ab-/P 67 kO/IFTJ 47 kD/P tn-2)

Fig. I. Neutrophil functions and eytoehrome b558 content of study patients on diiysO (D) and 180(11). (a): NBT test after stimuliUion with OPZ. Results are expressed as percentage of formazan-positive eells; (b) superoxide production after stimulation with PMA -t-fMLP; (c) cyiochrome b558 content of neutrophils In pmole/10" cells; (d) killing of S. aureus after 2 h ineubation (percentage of inoculum killed). Xb-/P: palients with X-linked cytochrome b558 detieiency; plaeebo treated, Ab/P: palients with a; r cytochrome b588 deficiency; placebo treated. 67-kD, IFN: palienl with fi7-kD protein deficiency; rlFN-/-treated; 47 kD/P: palient with 47-kD protein deficiency; placebo treated.

25-f"

20-

67kD/IFN

47 kO/P

Fig. 2. Total serum IgG (day 0, ^ ; day 180, • ) , IgGl (day 0. D; day 180, m) and IgG2 (day 0, • ; day 180, • ) levels of study patients. Abbreviations as in Fig. I.

T. J. Miihlebaeh et al.

206 (a)

(b)

A2urocidin +riFN>-

p29b

REFERENCES

-rlFN/

100

100-V

50

0 Cc)

90 0 CarbepanG

90

° 0 (d)

90 0

90

Lactofernn

100

50

Fig. 3. AP levels measured by densitometry of Western blots are shown for each subject, with day 0 and day 90 values connected by lines. Solid lines denote result,^ from one subject. Dashed lines are superimposable results for two dilVercnt subjects. Dotted lines arc superiinpositble results for three dilTereni subjects. Results arc expressed as percentage of simultaneously determined values for tour healthy donors, which varied by less than $"'