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Primary phagocytic cells are resistant to retrovirus mediated gene transfer for reasons outlined previously. EBV-immortalised B lymphocytes express all components of the NADPH-oxidase, and produce measurable quantities of superoxide following stimulation by agents such as PMA. Cells derived from patients with CGD mirror the functional defect of primary phagocytic cells, and are therefore useful models for reconstitution of the NADPH-oxidase activity by gene transfer.

The most sensitive and specific methods for detection of NADPH-oxidase activity are based on chemiluminescence. SOD-inhibitable direct reduction of ferricytochrome c has the advantage o f being quantitative, but is less sensitive, and can be misleading at low levels of activity. For immortalised B cells, which at best produce a small percentage of the superoxide generated by phagocytic cells, detection methods based on chemiluminescence are preferable. Similarly, because existing methods for cell-free determination of NADPH-oxidase activity were based on reduction o f ferricytochrome c, a heterologous chemiluminescence-based cell-free system was developed for the detection o f low levels o f cytosolic activity. This assay has the advantage of heightened sensitivity, and utilises at least 10-fold less cellular material than conventional assays.

Of all chemiluminescence-based assays for NADPH-oxidase function, peroxidase- catalysed oxidation of luminol to chemically excited 3-aminophthalate anion is most sensitive. This reaction is mediated by H2O2 formed by virtually instantaneous

B c 3 O O SDS40 SDS60 SDS80 SD S120 0.0 100 200

Time after activation (secs)

300 400

Fig 4.4. Optimisation of chemiluminescence-based detection for products of cell- free NADPH-oxidase activity. C>1osol and m em brane fractions purified from hum an neutrophils w ere prepared as described in m aterials and m ethods. For activation, 2 p g o f solu b ilised m em brane protein w as m ixed w ith 30fo,g o f c>1osol in the presence o f varying concentrations o f SD S (4 0 -1 2 0 p M ), and incubated at room tem perature for 2 m inutes before addition o f assay buffer (6 5 m M sodium phosphate buffer, pH 7.0, IniM E G T A , Im M M g C b , lO pM F A D , lO pM lu m in ol, lO U /m l horse radish peroxidase) to a total volum e o f lOOpl, and finally N A D P H (0 .2 m M ) to initiate the reaction. R eactions w ere incubated at 37®C, and all solutions pre-warm ed. M axim al activity w as ach ieved at concentrations o f S D S betw een 80 and 120p.M, measured on a lum inom eter.

[A]

[B]

T im e afte r activation (secs)

—■— p67.B1 □ p67.B2 -V - p47.B # p67.N X p 21rac 5 10 15 20

recom binant protein (m eg)

25

Fig 4.5 Comparison of neutrophil and B cell cytosolic activity in a cell-free assay, and enhancement by exogenous recombinant p67^*''^ protein. [A] C ytosol prepared from norm al im m ortalised B c e lls (B cyt), and cells derived from a patient w ith p47^*‘“ -d eficien t CGD (B cytC G D ) w ere tested for com plem entation and activation o f neutrophil m em branes in the ch em ilu m in escen ce-b ased cell-free system (see fig 4 .4 ). C onsistently, cytosol from neutrophils (PM N cyt) w as m ore active than that derived from B cells. M em branes alon e (P M N m em ) w ere inactive. [B] T h is observation is confirm ed in a conventional quantitative assay for production o f O2' by cytochrom e c reduction. For this reaction, 15pg o f solubilised m em branes w ere pre-m ixed w ith 2 0 0 p g cytosolic protein for 2 m inutes at room tem perature before addition o f assay buffer ( 6 5 m M sodium phosphate buffer, p H 7.0, Im M E G T A , Im M M gC b , lOpM F A D , lOOpM cytochrom e c to a total v o lu m e o f 1ml, and fin ally N A D P H (0.2m M ) to initiate the reaction, w h ich w as incubated at 37°C. SD S concentration w as optim ised for m axim al activity (~ 1 0 0 p M ). Superoxide production w as m easured for 9 0 s in a double-beam spectrophotom eter (U vikon 860, K ontron). T he reference sam ple w as identical, but w as supplem ented w ith superoxide dism utase (SO D 5 0 p g /m l). S O D -inhibitable cytochrom e c reduction w as estim ated by differential absorbance at 550n m and 55 7 n m (isob estic points). T he reaction w as supplem ented w ith affinity purified recom binant p47^*‘", p67^^" or p 2 1 ra cl pre-loaded w ith GTP (incubated w ith lOOpM GTP in 2m M E D T A for lO m ins, fo llo w ed by addition o f lO m M M gC lz). A ddition o f recom binant p67^*"^ enhanced activity o f both neutrophil and B cell cytosol In contrast, recom binant p47^*"* and p 2 1 ra cl produced no effect. T he absorption co -efficien t (e) for reduced cylochrom e c at 550n m w as taken as 2 1 .Im M "'.cm "'.

dismutation of O2 . To optimise conditions for a cell-free assay based on this reaction, activity was initially titrated using a cytosolic fraction and a particulate fraction enriched in membranes prepared from human neutrophils. Horseradish peroxidase (HRP) was added to excess, and activation initiated by addition of NADPH. The concentration of SDS in the cell free system is critical for maximal activity to be obtained, and was therefore optimised for each reaction (Fig 4.4). Subsequently, cytosolic extracts prepared from immortalised B cells were mixed with neutrophil membrane fractions. This resulted in optimised maximal activity similar, but consistently less than that using neutrophil cytosol (Fig 4.5A). In contrast, cytosolic fractions derived from patients with p47^^"^-deficient cells were completely inactive. These observations were confirmed in a conventional cell-free assay based on quantitative reduction of ferricytochrome c. Furthermore, supplementation of normal immortalised B cell cytosolic fractions with purified recombinant p67^^"^ restored activity to that of neutrophil cytosol (fig 4.5B). In contrast, addition of recombinant p47^^°^ or GTP pre-loaded (active) p2\rac\ had no effect. Supplementation of neutrophil cytosolic fractions with recombinant p67^^®^ also enhanced activity, indicating this to be the limiting cytosolic component at least in the cell-free system. In support of these findings. Western blot analysis o f B cell cytosolic fractions detected similar levels of p47^^^^ to that of neutrophils, but diminished levels of p67^^°^ (Chetty et al. 1995). Low levels of flavocytochrome in immortalised B cell membranes failed to support significant activity in either assay.

Development of a more sensitive cell-free assay for cytosol-based activity of the NADPH-oxidase is of genuine practical use for exploration of reconstitution methods, and may also provide a clearer insight into the mechanisms of action of this enzyme system in both immortalised B cells and primary phagocytes.