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The Journal of Immunology, 1998, 161: 4968-4974.
Copyright © 1998 by The American Association of Immunologists

Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Assembly and Activation in EBV-Transformed B Lymphoblastoid Cell Lines of Normal and Chronic Granulomatous Disease Patients1

Stefano Dusi2,*, Katyuscia A. Nadalini*, Marta Donini*, Lorena Zentilin{dagger}, Frans B. Wientjes{ddagger}, Dirk Roos§, Mauro Giacca{dagger} and Filippo Rossi*

* Institute of General Pathology, University of Verona, Verona, Italy; and {dagger} International Center for Genetic Engineering and Biotechnology, Trieste, Italy; {ddagger} Department of Medicine, University College London, Rayne Institute, London, United Kingdom; and § Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The NADPH oxidase is the enzymatic system responsible for the generation of bactericidal oxygen free radicals in phagocytes (1, 2, 3, 4, 5, 6, 7, 8, 9). It is composed of several proteins, some located on specific granules, secretory vescicles, and plasma membrane, i.e., the cytochrome b558, and others in the cytosol, i.e., p47phox, p67phox, p40phox, and the small G proteins, Rac-1 and Rac-2 (1, 2, 3, 4, 5, 6, 7, 8).

The activation of NADPH oxidase involves the translocation of cytosolic components p47phox, p67phox, p40phox, and Rac G proteins to the plasma membrane, where they associate with cytochrome b558, forming an electron transport chain responsible for the reduction of molecular oxygen to superoxide anion (1, 2, 3, 4, 5, 6, 7, 8). Despite the progress obtained from study of the mechanisms of NADPH oxidase assembly (reviewed in Refs. 2–8), the exact role played by each component as well as the interactions occurring between the cytosolic and the membrane factors remain to be elucidated. It has been demonstrated that the activation of NADPH oxidase is associated with phosphorylation of the cytosolic components (9, 10, 11, 12, 13), and that the translocation of p67phox and p40phox is facilitated by p47phox (14, 15, 16), which binds proline-rich regions on p22phox (17, 18, 19, 20) and multiple sites on gp91phox (21).

Recently, generation of moderate amounts of superoxide anion by several nonphagocytic cell types, including endothelial cells (22), fibroblasts (23, 24), mesangial cells (25, 26), chondrocytes (27), and smooth muscle cells (28), has been described. NADPH oxidase-like enzymes have been suggested as the source of these radicals, since components of the NADPH oxidase system have been detected in many cases (24, 26, 27, 28, 29). The physiologic role of NADPH oxidase-like enzymes in nonphagocytic cells remains unknown. However, the low amounts of reactive oxygen species produced by these enzymes do not seem to have a microbicidal role, but could act as signaling molecules, influencing many cell functions (30).

B lymphocytes also produce superoxide anion upon stimulation with various agonists (31, 32, 33, 34, 35, 36, 37, 38). The electron transport chain expressed in B lymphocytes seems to be structurally homologous, if not identical, with the NADPH oxidase of phagocytes (37, 39, 40, 41, 42, 43, 44, 45, 46, 47).

With regard to the mechanisms of NADPH oxidase assembly, previous experiments performed with polymorphonuclear neutrophils (PMN)3 from patients affected by X-linked chronic granulomatous disease (CGD) (31) have demonstrated that the presence of cytochrome b558 is required for translocation of the cytosolic components of NADPH oxidase to the membranes (14). However, such experiments did not distinguish the role of the single gp91phox and p22phox subunits of cytochrome b558 in translocation of the cytosolic components.

In this paper we investigate this question in detail. For this purpose we used EBV-transformed human B lymphoblastoid cell lines (B cells) from X-linked CGD patients expressing p22phox in the absence of gp91phox. The results demonstrate that in these cells, p47phox, p67phox, and p40phox do not translocate to the membranes upon stimulation with PMA, indicating that gp91phox is required for the binding of cytosolic components to cytochrome b558. We also used B cells from an X91+ CGD patient in whom p22phox was normally expressed and gp91phox was present but lacked five amino acids. In these cells, while translocation of p47phox was unaffected, p67phox and p40phox were very poorly translocated, indicating that p47phox can associate with the membranes independently of p67phox and p40phox, and that gp91phox could have a specific role in the binding of p67phox and p40phox to the membranes. Because the production of O2- in response to PMA was greatly reduced in the B cells of this patient, our finding also suggests that the translocation of p67phox and p40phox might represent the limiting factor for the activation of NADPH oxidase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PMA was purchased from Sigma (St. Louis, MO). SDS, acrylamide, N,N'-methylenbisacrylamide, tetramethylenediamine, and blotting nitrocellulose membranes were purchased from Bio-Rad (Richmond, CA). Ficoll 400 and m.w. standards were obtained from Amersham (Aylesbury, U.K.). Anti-p67phox, anti-p47phox, and anti-p40phox rabbit polyclonal antisera were gifts from Dr. A. W. Segal (Department of Medicine, University College, London, U.K.) (48, 49, 50). Anti-gp91phox and anti-p22phox mouse mAbs were obtained from the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands) (51).

Isolation of neutrophils

Human neutrophils were prepared from venous blood of healthy donors as previously described (52), washed, and resuspended in HBSS buffered with 20 mM HEPES (pH 7.4) and containing 0.5 mM CaCl2 and 5.6 mM glucose.

Establishment of B lymphoblastoid cell lines

Mononuclear cells were isolated from 10 ml of the heparinized blood samples from affected individuals by centrifugation over Ficoll-Hypaque density gradients. B lymphoblastoid cell lines were established by infection with EBV obtained from the supernatant of the B95-8 marmoset cell line following previously described procedures (53). Briefly, total mononuclear cells were resuspended in the supernatant of B95-8 cells diluted 1/1 with fresh medium at a cell concentration of 2 x 106/ml. Half the virus-containing medium was replaced, at the latest, 24 h after starting the culture by addition of RPMI 1640 with 20% FCS and 2 µg/ml (final concentration) of cyclosporin A (Sandimmum, Sandoz, Hanover, NJ). The medium was then refreshed once a week by removing half the supernatant and replacing it by fresh medium containing 1 µg/ml of cyclosporin A.

Characterization of the genetic defect

The coding region of the gp91phox mRNA was reverse transcribed from 1 µg of total RNA extracted from B lymphoblastoid cell lines from the patients and amplified by PCR using the following three primer pairs: CGD 1 (20 nucleotides), 5'-TGAATGAGGGGCTCTCCATT-3'; CGD B (26 nucleotides), 5'-GTACAATTCGTTCAGCTCCATGGATTG-3'; CGD A (25 nucleotides), 5'-CCGGAGGTCTTACTTTGAAGTCTTT-3'; CGD DII (20 nucleotides), 5'-GCAAACCACTCAAAGGCATG-3'; CGD C (25 nucleotides), 5'-GGTGATGTTAGTGGGAGCAGGGATT-3'; and CGD 8 (20 nucleotides), 5'-GTAAAAGTGCTCTCAAAACC-3'.

Three overlapping fragments were obtained for each patient. Conditions for RT were 10-min annealing at 65°C and 1-h extension at 37°C, followed by 5 min at 95°C. Conditions for PCR amplifications were 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C, followed by 5 min of final extension at 72°C.

To analyze the very 5' portion that encompasses the initiator ATG codon, genomic DNA was amplified using primers in the 5'-untranslated region and in the first intron region, respectively (primers CGD 11 (20 nucleotides), 5'-GCATAGTATAGAAGAAAGGC-3'; CGD 12 (18 nucleotides), 5'-TGGTACTTACAATGACAA-3').

PCR was performed in a Perkin-Elmer thermal cycler (Norwalk, CT) according to standard procedure. The coding region of the gp91phox cDNA was evaluated by direct sequencing of the amplified fragments with a commercial kit (AmpliCycle sequencing kit, Perkin-Elmer).

Superoxide anion production

Superoxide anion (O2-) release from PMN and B cells was estimated by superoxide dismutase-inhibitable cytochrome c reduction (54). Briefly, the cells were placed in 96-well plates (2 x 105 cells/well in a total volume of 200 µl of HBSS plus 10% FCS, pH 7.4, containing 80 µM ferricytochrome c type III; Sigma) with or without 300 U/ml superoxide dismutase (Sigma) and in the presence or the absence of 100 ng/ml PMA (Sigma). The reduction of cytochrome c was read at 550 nm.

Preparation of lysates and membranes of B lymphocytes and PMN

Human neutrophils and B cells (1 x 107/ml) were treated with PMA for various lengths of time at 37°C in a water bath under continuous shaking. Reactions were stopped by diluting the cells with a 10-fold excess of ice-cold HBSS containing 1 mM PMSF, 2 mM sodium orthovanadate (Na3VO4), and 10 µM phenylarsine oxide. To prepare whole cell lysates, the cells were pelleted, resuspended in electrophoresis sample buffer (60 mM Tris-HCl, 20% (v/v) glycerol, 4% (w/v) SDS, and 2% (v/v) 2-ME, pH 6.8), and boiled for 5 min at 100°C. For preparation of the membranes, PMN or B cells were centrifuged and resuspended in 0.4 ml of ice-cold relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and 10 mM PIPES, pH 7.3) containing 5 µg/ml leupeptin, 5 µg/ml pepstatin, 10 µM phenylarsine oxide, 2 mM sodium orthovanadate, 1 mM PMSF, 5 mM sodium fluoride, and 1 mM di-isopropylfluorophosphate (all from Sigma). Cells were then disrupted by two cycles of sonication for 30 s each at 100 W with a Labsonic 1510 sonicator (B. Braun, Melsungen, Germany). Intact cells and nuclei were pelleted by centrifugation at 500 x g for 10 min at 4°C. The supernatant was carefully layered on top of a discontinuous sucrose gradient prepared with 1.5 ml of 15% sucrose and 1.5 ml of 34% (w/w) sucrose made up in the relaxation buffer and was centrifuged at 100,000 g for 40 min in a Beckman L5–50B ultracentrifuge using an SW50 rotor (Beckman, Palo Alto, CA). The light membrane fractions were collected at the 15/34% interface, washed in cold relaxation buffer, and pelleted by ultracentrifugation at 100,000 x g for 30 min. Membranes were then resuspended in electrophoresis sample buffer and boiled for 5 min at 100°C.

Electrophoresis and immunoblotting

After measurement of the protein content of each sample, aliquots of cell lysates or membranes containing equivalent amounts of protein were subjected to SDS-PAGE on 12% gels. Proteins were transferred from the gels to nitrocellulose membranes (Bio-Rad) in a Bio-Rad Trans Blot Cell apparatus. Blotting was performed at 70 V for 90 min in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, and 3.5 mM SDS, pH 8.3, at 4°C. To ensure that comparable amounts of proteins had been transferred to the nitrocellulose membranes, proteins were revealed on the nitrocellulose membranes by staining with 0.02% (v/v) Ponceau S (Sigma) for 1 min. The blots were then rinsed in TBS (50 mM Tris, 170 mM NaCl, and 0.2% (v/v) Tween-20, pH 7.5) and incubated for 90 min in TBS containing 5% BSA (blocking buffer), before incubation overnight at 4°C with rabbit anti-p67phox, anti-p47phox, anti-p40phox, or mouse anti-gp91phox and anti-p22phox diluted 1/500 in TBS containing 1 mg/ml BSA. The blots were rinsed with several changes of TBS and then incubated for 120 min at room temperature in horseradish peroxidase-labeled donkey anti-rabbit IgG or goat anti-mouse IgG (both from Amersham) diluted 1/15,000 and 1/2,000, respectively, in TBS containing 1 mg/ml BSA. After further washing, bound Abs were detected by enhanced chemiluminescence Western blotting detection reagents (Amersham). Multiple exposures of the same blot were performed to ascertain that the enhance chemiluminescence signal was in the linear range of sensitivity. When required, the blots were stripped for 30 min at 50°C in a solution containing 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The entity of the respiratory burst and the expression of NADPH oxidase components in B cells

To understand which is the molecular basis of the low respiratory burst observed in B cells, we compared the expression of the different NADPH oxidase components in B cells and in PMN. Figure 1Go shows that the NADPH oxidase components p67phox, p47phox, p40phox, gp91phox, and p22phox were all detectable in B cells. The levels of p47phox expression in PMN and in B cells were very similar, while the amounts of p67phox, p40phox, and the subunits of cytochrome b558, p22phox and gp91phox, were reduced in B cells compared with PMN, the main deficit regarding the cytochrome b558 components (Fig. 1Go). A semiquantitative densitometric analysis of the bands showed that, in comparison with PMN, the amounts of p47phox, p67phox, p40phox, and p22phox in B cells were reduced by about 6, 70, 38, and 96%, respectively. The densitometric analysis of gp91phox was not possible because it was hampered by the heavy glycosylation of this protein (46). While in PMN, Rac-1 and Rac-2 were recognized by specific Abs, attempts to detect these proteins in B cells did not produce reliable results (data not shown). This could be due to the low level of expression of Rac G proteins in B cells. Data reported by others regarding the expression of Rac-1 and Rac-2 proteins in B cells are contradictory (46, 55, 56).



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  		FIGURE 1. Expression of NADPH oxidase components in normal B cells and PMN. Neutrophils (PMN) and EBV-transformed B cell lines (B cells) from two healthy, unrelated donors (1 and 2) were pelleted and immediately boiled in electrophoresis sample buffer. Aliquots of cell lysates (30 µg/lane) were subjected to SDS-12% PAGE and immunoblotted with Abs raised against the indicated components of NADPH oxidase. The data are from one experiment representative of two performed.

 
Expression of gp91phox and p22phox subunits of cytochrome b558 in B cells from X-linked CGD patients

During studies concerning the characterization of the mutations that underlie several cases of X-linked CGD, we selected eight patients affected by X-linked CGD. B cells from seven of these patients did not produce O2- in response to PMA, while B cells from one patient (M.C.) produced low amounts of O2-, corresponding to 20% of those observed in normal cells (Table IGo). We then identified the mutations responsible for the disease (Table IGo).


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  		Table I. Mutations in X-CGD patientsa

 
It is generally known that in PMN from X-linked CGD patients, the absence of gp91phox is accompanied by the lack of p22phox (31). We investigated the expression of gp91phox and p22phox in lysates of PMN and B cells from three unrelated X-linked CGD patients (G.T., D.F., and L.C.) through immunoblotting with anti-gp91phox and anti-p22phox Abs. The data reported in Figure 2Go show that while in PMN of the patients in the absence of gp91phox, p22phox was also absent (or, in some cases, present in vanishing amounts), in B cells of the same patients p22phox was normally expressed despite the lack of gp91phox. To ascertain whether the presence of p22phox represents a general characteristic of B cells of X-linked CGD, we investigated the expression of NADPH oxidase components in B cells from the other four patients (A.Z., N.V., E.O., and N.B.), whose PMN were not available. Figure 3Go shows that also in B cells of these patients, only gp91phox was absent, while p22phox was normally expressed. This finding seems to indicate that the different expressions of p22phox and gp91phox are characteristic of B lymphocytes in X-linked CGD.



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  		FIGURE 2. Expression of gp91phox and p22phox in PMN and B cells from X-linked CGD patients. Neutrophils (PMN) and EBV-transformed B cell lines (B cells) from healthy donors (normal) and three unrelated X-linked CGD patients (G.T., D.F., and L.C.) were boiled in electrophoresis sample buffer, and aliquots of cell lysates (20 µg/lane) were subjected to SDS-12% PAGE and immunoblotted with anti-gp91phox and anti-p22phox Abs. The band of about 100 kDa recognized by the anti-gp91phox Ab in all B cell lanes does not represent gp91phox but, rather, a cross-reactive protein, as previously stated by Maly et al. (20). The data are from one experiment that was repeated three times.

 


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  		FIGURE 3. Expression of NADPH oxidase cytosolic and membrane components in B cells from X-linked CGD patients. EBV-transformed B cell lines from an healthy donor (normal), five patients affected by classical X-linked CGD (X91°; A.Z., N.V., E.O., N.B., and D.F.), and one patient with a gp91phox-positive form of CGD (X91+; M.C.) were pelleted and boiled in electrophoresis sample buffer; aliquots of cell lysates (20 µg/lane) were subjected to SDS-12% PAGE and immunoblotted with Abs raised against the indicated components of NADPH oxidase. The band of about 100 kDa recognized by the anti-gp91phox Ab in all B cell lines does not represent gp91phox but, rather, a cross-reactive protein (20). The data are from one experiment that was repeated three times.

 
Roles of gp91phox and p22phox in the translocation of the cytosolic components of NADPH oxidase

It has been shown that in CGD PMN, in which both subunits of cytochrome b558 are lacking, p47phox, p67phox, and p40phox do not translocate to the membranes (14). This finding, which indicates that cytochrome b558 is required for association of the cytosolic components with the membranes, does not allow distinction between the single subunits of cytochrome b558 in this process. The availability of CGD B cells expressing p22phox in the absence of gp91phox prompted us to investigate the role of each subunit of cytochrome b558 in the translocation of NADPH oxidase cytosolic components. Figure 3Go shows that, as expected, the cytosolic components of the NADPH oxidase, p67phox, p47phox, and p40phox, were normally expressed in the B cells of the X-linked CGD patients. Upon stimulation of B cells from normal subjects with PMA, p67phox, p47phox, and p40phox translocated to the membranes (Fig. 4Go), indicating that the activation of NADPH oxidase in B lymphocytes involves the assembly of components of the enzyme on the membranes, as occurs in PMN. By using specific anti-cytochrome b558 Abs, we demonstrate that while p22phox was present on the light membrane fractions from B cells of CGD patients (Fig. 4Go), gp91phox was not detectable (data not shown). Figure 4Go also illustrates that in PMA-stimulated B cells from CGD patients expressing p22phox but not gp91phox, the cytosolic components p47phox, p67phox, and p40phox failed to associate with the membranes. This result indicates that p22phox alone does not allow association of cytosolic components with the membranes.



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  		FIGURE 4. Translocation of p67phox, p47phox, and p40phox from cytosol to membranes of X-linked CGD B cells expressing p22phox in the absence of gp91phox. Neutrophils from a healthy donor (PMN) and EBV-transformed B cell lines (B cells) from a healthy subject (normal) and from three X-linked CGD patients expressing p22phox in the absence of gp91phox (A.Z., N.V., and E.O.) were incubated at 37°C under agitation in the absence or the presence of PMA (100 ng/ml). Owing to the different kinetics of the NADPH oxidase responses in the two cell types, the reactions were stopped at 5 min for PMN and at 45 min for B cells. The cells were then sonicated and fractionated through a sucrose gradient (see Materials and Methods). Aliquots of membrane fractions from resting (-) and stimulated (+) cells containing equal amounts of protein were subjected to SDS-12% PAGE and immunoblotting with anti-p67phox, anti-p47phox, anti-p40phox, and anti-p22phox Abs. The data are from one experiment representative of three performed.

 
Translocation of NADPH oxidase components in B cells from a patient affected by X91+ form of CGD

Patient M.C. was affected by a particular form of CGD, which allowed us to better investigate some aspects of the process of NADPH oxidase assembly. The B cells of this patient showed a very little O2- production in response to PMA (0.22 nmol/30 min/2 x 105 M.C. cells and 1.12 nmol/30 min/2 x 105 normal B cells) despite the normal presence of all NADPH oxidase components, including both subunits of cytochrome b558 p22phox and gp91phox (Figs. 3Go and 5Go). The genetic analysis of M.C. B cells showed the presence of a deletion affecting nucleotides 902 to 916 in the coding region of gp91phox, predicting the lack of amino acids 298 to 302 in the gp91phox protein (Table IGo). Thus, MC was affected by a rare form of X-linked CGD (X91+) in which cytochrome b558 was present but was mutated and not fully functional. Because cytochrome b558 is essential for the assembly and activation of NADPH oxidase, we wondered how the mutation on gp91phox affected activation of the NADPH oxidase of the M.C. B cells. To answer this question we examined the translocation of the cytosolic components of NADPH oxidase in M.C. B cells treated with PMA, and we found that the mutation affecting gp91phox produced a differentiated translocation of NADPH oxidase cytosolic components. In fact, as shown in Figure 5Go, in the B cells of M.C., while p47phox was translocated as in normal cells, the association of p67phox and p40phox with the membranes was greatly depressed. This finding indicates that the deletion of amino acids 298 to 302 on gp91phox did not modify the translocation of p47phox, but decreased that of p67phox and p40phox. This means that p47phox can translocate to the membranes independently of p67phox and p40phox, and that gp91phox might mediate and/or stabilize the association of p67phox and p40phox with the membranes.



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  		FIGURE 5. Translocation of p67phox, p47phox, and p40phox to the membranes of B cells from a patient affected by X-linked gp91phox-positive CGD. EBV-transformed B cell lines from a healthy donor (normal) and from a gp91phox-positive patient (M.C.) were stimulated with 100 ng/ml PMA at 37°C under agitation for 30 min. The reaction was stopped, and the cells were fractionated as described in Figure 4Go. Resting (-) and stimulated (+) membranes were subjected to SDS-12% PAGE and immunoblotted with Abs raised against p67phox, p47phox, and p40phox. The blot was then stripped and reprobed with anti-gp91phox and anti-p22phox Abs. The data are from one experiment that was repeated three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work we addressed the mechanisms of activation of NADPH oxidase, a process dependent on the assembly of membrane and cytosolic components, in immortalized B lymphoblastoid cell lines of X-linked CGD patients expressing the two subunits of the cytochrome b558 in a fashion different from that of PMN.

Before studying the translocation of cytosolic components to the membranes in cells from CGD patients, we investigated the expression and assembly of components of NADPH oxidase and the entity of activation of this enzyme in normal B cells. First, we confirmed previous findings (46) that in B cells the expression of p47phox attained a level more or less equivalent with that found in PMN, while those of p40phox and, in particular, p67phox, gp91phox, and p22phox were lower in B cells than in PMN. The pattern of this expression must represent the main molecular basis of the low production of O2- in B cells with respect that in PMN (32, 33, 34, 35, 36, 37, 38, 39, 46).

The second point we investigated was translocation of the cytosolic components, and the results have shown that in normal B cells the stimulation with PMA induces a translocation of p67phox, p47phox, and p40phox from cytosol to the membranes as occurs in PMN. To our knowledge, this is the first demonstration that p67phox and p40phox associate with the membranes in stimulated B cells.

At this point we started the investigations on NADPH oxidase of B cells from CGD patients. It is generally known that in PMN from patients affected by the X-linked form of CGD, the absence of gp91phox, due to a mutation in the gene for gp91phox, is accompanied by the lack of expression of p22phox despite the normality of its gene (9). It has been reported by Chetty et al. that in B lymphoblastoid cell lines from two patients with the X-linked form of CGD, both p22phox and gp91phox were lacking (46). However, other authors have observed that p22phox was detectable in B cells of subjects with X-linked CGD in the absence of gp91phox (42, 44, 57). Here we confirm that while in PMN of subjects affected by X-linked CGD, p22phox was lacking, in B cells from seven unrelated X-linked CGD patients, p22phox was always expressed despite the absence of gp91phox. We also show that the single p22phox is present in the light membrane fraction of B cells from X-linked CGD patients. This finding suggests that gp91phox is not required for the post-translational association of p22phox with the membranes. The fact that in B cell lines of two patients with X-linked CGD, p22phox has not been detected (46) might be explained on the basis of the different procedures used to process the cells.

It is widely accepted that cytochrome b558 is required for translocation of the cytosolic components of NADPH oxidase to the membranes. This concept derives from experiments performed on PMN of CGD patients in which both subunits of cytochrome b558 were lacking (14). However, these experiments did not permit a distinction between the role of the single gp91phox and that of p22phox subunit in the translocation of the cytosolic components. By means of several other experimental approaches it has been shown that p47phox can bind both p22phox and gp91phox subunits of cytochrome b558 (17, 18, 19, 20, 21). However, leukocytes expressing only one subunit of cytochrome b558 in the absence of the other have never been used to investigate the roles of gp91phox and p22phox in translocation of the cytosolic components of NADPH oxidase. The availability of CGD B cells expressing p22phox in the absence of gp91phox prompted us to address this problem by investigating the translocation of p47phox, p67phox, and p40phox in these cells. The results have shown that upon stimulation with PMA, in such cells the translocation of p47phox, p67phox, and p40phox does not take place. This finding suggests that p47phox cannot interact with the single p22phox. This could be due to the fact that in the absence of gp91phox, p22phox can assume an altered conformation in the membrane, so that the binding sites for p47phox are no longer accessible. Alternatively, the conformation of p22phox is not changed in the absence of gp91phox, but the presence of gp91phox is required to mediate and/or stabilize the interaction between p47phox and p22phox. These results are in agreement with the previous finding that a recombinant 32S-labeled p47phox failed to recognize the single subunits of the cytochrome b558 immobilized on nitrocellulose membranes (58) and that in a case of CGD in which the cytochrome b558 was present but not functional because of a point mutation affecting gp91phox, p47phox and p67phox did not translocate to the membranes despite the normality of p22phox (59). In contrast, our results are not consistent with those of Leto et al. (60), who showed that in K562 erythroleukemia cells cotransfected with p22phox and p47phox, the latter protein was able to associate with the membranes. This discrepancy might be due to the particular experimental conditions in which the translocation has been studied by these authors.

It has been demonstrated that p47phox translocates to the membranes as a free form or as a complex with p67phox and p40phox, while p67phox and p40phox translocate only as a complex with p47phox and do not exist as free forms in the cell (61, 62, 63, 64). Here we report that in B cells from a patient who carries a rare form of CGD in which the gp91phox is present but lacks amino acids 298 to 302 while translocation of p47phox to the membranes is unaffected, p67phox and p40phox were very poorly translocated. This finding clearly demonstrates that upon cell stimulation, the aliquot of cytosolic p47phox that is not complexed with p67phox and p40phox can translocate to the membranes as previously suggested (61, 64), and the mutation in gp91phox prevents association of the complex p47phox/p67phox/p40phox with the membranes despite the fact that the deletion did not affect the sites involved in the binding of p47phox to cytochrome b558 (7, 8). Binding sites for p47phox have been found on p22phox and gp91phox (17, 18, 19, 20, 21). However, recent studies have suggested that p67phox could also interact directly with cytochrome b558 (8, 65, 66). Therefore, the deletion affecting gp91phox could modify some sites on this protein involved in interactions with the complexed p47phox and/or p67phox, leading to a depression of association of the complex p47phox/p67phox/p40phox to the membranes. It cannot be ruled out that the site containing the amino acids deleted in gp91phox of M.C. belongs to a binding site for p67phox.

Experiments performed by Cross et al. to clarify the roles of p67phox and p47phox in the regulation of electron flow in NADPH oxidase have shown that p67phox mediates electron flow from NADPH to FAD, whereas p47phox mediates electron flow from FAD to heme and then to oxygen (67). This indicates a distinct function for p67phox and p47phox, probably mediated by separate binding of these components to gp91phox. On this basis, it can be postulated that the low production of O2- in B cells of M.C. is due to a slow electron flow from the NADPH to flavin center of cytochrome b558 because of a slight translocation of p67phox.

In activated B cells of M.C., i.e., under conditions in which the association of p67phox and p40phox with the membranes was greatly reduced while that of p47phox was unaffected, the respiratory burst was very depressed. This is in agreement with the concept that the limiting factor for the activation of NADPH oxidase is p67phox. In fact, Koshkin et al. (68) found that in a cell-free system, pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b558 to p67phox and Rac-1 in the total absence of p47phox, while combinations of two cytosolic components other than p67phox and Rac-1 were incapable of activation. Therefore, our data could be in agreement with the conclusions drawn by Koshkin et al. (68) and by Freeman et al. (66) that p67phox together with Rac could be the cytosolic components directly involved in the induction of electron transport in cytochrome b558, while p47phox would have a role in facilitating or stabilizing the interactions of p67phox, p40phox, and Rac with cytochrome b558. However, it cannot be ruled out that the mutation observed in M.C. may also induce a conformational change in gp91phox, affecting its ability to transport electrons independently of its interactions with the cytosolic components.

In conclusion, by using CGD lymphocytes it has been possible to demonstrate that 1) neither the free form of p47phox (i.e., the aliquot of p47phox that is not complexed with p67phox and p40phox) nor the p47phox/p67phox/p40phox complex can bind to p22phox on the membranes in the absence of gp91phox; 2) the free form of p47phox can associate with cytochrome b558 independently of the complex p47phox/p67phox/p40phox; and 3) gp91phox may have a role in mediating and/or stabilizing binding of the p47phox/p67phox/p40phox complex to the membranes.


   Footnotes
 
1 This work was supported by grants from Consiglio Nazionale delle Ricerche (no. 97.04259.CT04), from Cofinanziamento Ministero dell’Universitá e della Ricerca Scientifica e Tecnologica e Università di Verona del Programma di Ricerca: Infiammazione: Biologia e Clinica, from Giunta Regionale Veneto-Ricerca Sanitaria Finalizzata-Venezia, and Progetto Sanità 1996/97, Fondazione Cassa di Risparmio di Verona, Vicenza, Belluno, and Ancona. Back

2 Address correspondence and reprint requests to Dr. Stefano Dusi, Istituto di Patologia Generale, Strada Le Grazie, 37134 Verona, Italy. Back

3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; CGD, chronic granulomatous disease; gp91phox, p22phox, p47phox, p67phox, and p40phox, phagocyte oxidase factors of 91, 22, 67, 47, and 40 kDa, respectively. Back

Received for publication January 27, 1998. Accepted for publication June 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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