Mechanisms involved in the deposition of immune complexes in tissues.

The mechanisms reponsible for the deposition of circulating immune complexes have been analyzed. An active process appears to be responsible in both a laboratory model in guinea pigs and in acute immune complex disease (serum sickness) in rabbits. In rabbits, after the injection of antigen to induce serum sickness, immune complexes appear in the circulation. In addition, homocytotropic (IgE) antibody is formed which binds to the surface of basophils. Leukocyte suspensions containing these basophils, when combined with specific antigen, release a soluble factor that causes clumping of platelets and release of their vasoactive amines. An excellent correlation was found between the presence of this mechanism of release of vasoactive amine and the deposition of immune complexes in serum sickness of rabbits. Antagonists of vasoactive amines or depletion of platelets, the major circulating reservoir of these amines, suppressed the deposition of circulating immune complexes and inhibited glomerulitis and arteritis. Upon entering the walls of vessels, the complexes became lodged immune complexes, greater than 19S in size, were deposited along the membranes. The data suggest that at a time when immune complexes appear in the circulation of an immunized rabbit, vasoactive amines are released from platelets in areas where turbulence of blood occurs. Sensitized basophils participate in the release of vasoactive amines from the platelets. The amines induced increased vascular permeability which leads to deposition of large complexes from the circulation in vessel walls by a process of filtration. The deposited complexes then induce inflammatory injury.

Studies on circulating immune complexes. 3. Factors governing the ability of circulating complexes to localize in blood vessels.

The relationship between certain physicochemical properties of circulating immune complexes and their ability to localize in vessel walls during a state of increased permeability was studied. The ability to become deposited was related to the large size of complexes, rather than to their net charge or to a specific affinity between complexes and structures of vessel walls. Soluble complexes with sedimentation rates greater than 19S were capable of being entrapped along the vessel wall membranes, while complexes smaller than this were not. These large complexes were removed rapidly from the circulation, while smaller complexes persisted. Minimal levels of total complexes in the circulation necessary for detectable vascular localization were found to be as low as 15 microg antibody N/ml plasma. In experimental serum sickness, a disease known to be induced by circulating immune complexes, the development of vascular and glomerular lesions occurred almost exclusively in rabbits having large (greater than 19S) circulating immune complexes. Animals with smaller complexes did not show deposition of complexes in glomeruli or development of glomerulonephritis. Their incidence of vasculitis was markedly reduced.

Dissemination of contact activation in plasma by plasma kallikrein.

The dissemination of contact activation of plasma was examined by measuring the cleavage of Hageman factor (HF) molecules on two separate sets of kaolin particles, one of which contained all of the components of the contact activation system, HF, prekallikrein (PK) and high molecular weight kininogen (HMWK) in whole normal plasma, and the second set of particles containing only HF and HMWK, being prepared with PK-deficient plasma. After mixing of the particles, cleavage of HF on the second set of particles occurred at a rate similar to that occurring on the first set of particles. This indicated that rapid dissemination and burst of activity of the contact reaction takes place in fluid phase. A supernatant factor, responsibel for the dissemination of the contact reaction, was identified as kallikrein. A rapid appearance of cleaved PK (kallikrein) and HMWK on both the kaolin surface and in the supernate was observed. Within 40 s, > 70-80% of the PK and HMWK in the supernate was cleaved. On the surface, approximately 70% of each radiolabeled protein was cleaved at the earliest measurement. Cleavage of PK by activated HF occurred at least 17 times faster on the surface than in the fluid phase, as virtually no cleavage of PK occurred in fluid phase. Each molecule of surface-bound, activated HF was calculated to cleave at a minimum, 20 molecules of PK per minute. It is concluded that the contact activaton of plasma may be divided into three phases: (a) the reciprocal activation of a few molecules of zymogen HF and PK on the surface, with HMWK acting as cofactor to bring these molecules into apposition; (b) the rapid release of kallikrein into the fluid phase and the continued conversion of PK to kallikrein by each surface-bound molecule of activated HF; and (c) the activation by fluid-phase kallikrein of multiple surface-bound HF molecules, and the cleavage of multiple molecules of MHWK both in fluid phase and on the surface by the soluble kallikrein. The evidence suggests that steps b and c account for a great majority of the generation of contact activation of plasma.

The localization of circulating immune complexes in experimental serum sickness. The role of vasoactive amines and hydrodynamic forces.

In serum sickness, mechanisms by which circulating immune complexes become localized in the walls of vessels and glomeruli have been studied. In affected arteries, morphologic observations showed that circulating marker particles of carbon would rapidly deposit along the luminal surface of the internal elastic lamina. This, as in previous studies, suggested an increase in vascular permeability during which large molecules were capable of being trapped by a filtering membrane in the vessel wall. In attempts to prevent the increase in vascular permeability, rabbits were treated with antagonists of histamine and serotonin. Such treatment markedly inhibited the localization of immune complexes in glomeruli, the development of proteinuria, and glomerular endothelial proliferation. Cardiovascular lesions also were largely prevented from developing. Depletion of platelets, the principal reservoir of vasoactive amines, had a similar though less pronounced effect. While the deposition of immune complexes was inhibited, allergic inflammation in general was not, since normal rabbits treated as above were found capable of developing full Arthus reactions and acute nephrotoxic nephritis. Hydrodynamic factors were noted to be important in determining the location of arterial lesions. Studies of aortas from unmodified rabbits and from those with surgically induced coarctations of the abdominal aorta revealed intimal lesions concentrated at areas of high turbulence, such as at branches, bifurcations, outflows and zones of configurational change. Lesions in these areas were also largely inhibitable by depletion of platelets or by antagonists of histamine and serotonin.

Isolation and characterization of permeability factors from rabbit neutrophils.

Four basic proteins that increase vascular permeability have been isolated in purified form from rabbit neutrophilic granules. These proteins are termed band 1, 2, 3, and 4 protein according to their electrophoretic migration in acrylamide gel. Molecular weights of band 1 and 2 protein derived from amino acid composition were 4800 and 5300, respectively. These values are in good agreement with those obtained for these proteins by gel diffusion techniques. The molecular weight of band 3 protein was also in the range of 5000 by the latter technique. The molecular weight of band 4 protein determined by ultracentrifugal analysis and amino acid composition was 12,000. Although all four proteins had the capacity to induce immediate increase in vascular permeability, only band 2 protein was found to release histamine from isolated rat peritoneal mast cells. Furthermore, it has been shown that the permeability-inducing activity of band 2 protein can be inhibited by pretreating rabbits with antihistamine. Band 2 protein did not release histamine from rabbit platelets and depletion of rabbit platelets from the circulation had no influence on the permeability-inducing activity of this protein. Band 1, 3, and 4 proteins did not release histamine from isolated rat peritoneal mast cells and their capacity to increase vascular permeability remained unaffected by treatment of rabbits with antihistamine. These investigations suggest that the histamine-releasing activity of band 2 protein is a specific phenomenon and is associated with particular amino acid grouping or spacial configuration of the molecules. By the same token, the increase in vascular permeability induced by the nonhistamine-releasing band 1, 3, and 4 proteins represents a specific phenomenon (or phenomena) not particularly related to the over-all charge of these molecules.

The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement.

Anaphylatoxin activity was derived from both human C'5 and C'3 molecules. This was achieved in the case of C'5 by interaction with trypsin or with EAC'4, (oxy)2a, 3. The smooth muscle-contracting material obtained from the treated C'5 was found to be a fragment of approximately 9,000-11,000 molecular weight. Its action was inhibited with antihistamine. The trypsinized C'5 also increased vascular permeability in guinea pig skin. When human C'3 was incubated with C'3 inactivator complex, which consists of a cobra venom protein and a beta-globulin of human serum, anaphylatoxin activity was observed. The activity was associated with a fragment cleaved from the C'3 molecule, having a molecular weight of between 6,000 and 15,000 as determined by gel filtration techniques. Similar activity was derived from C'3 by the C'3-converting enzyme in free or in cell-bound form. The C'5 anaphylatoxin failed to cross-desensitize guinea pig ileum to the contracting capacities of C'3 and guinea pig anaphylatoxin and vice versa. Anaphylatoxin prepared from C'3 by all methods mentioned above caused cross-desensitization to the other C'3 derivatives, but failed to desensitize to guinea pig anaphylatoxin.

Polymorphonuclear leukocytes in immunologic reactions. The destruction of vascular basement membrane in vivo and in vitro.

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.

Immunological induction of increased vascular permeability. II. Two mechanisms of histamine release from rabbit platelets involving complement.

Two mechanisms have been described whereby rabbit platelets in vitro may be induced to release their contained histamine by the reaction of antigen and antibody. Both processes require the participation of the complement system. In the first, the adherence of platelets to particulate antigens such as zymosan or erythrocytes which have fixed complement through the third component was followed by histamine release. Plasma lacking C6 activity was fully active in this system. In the second mechanism, the reaction of soluble antigen with antibody in the presence of plasma also caused release of histamine from platelets and platelet clumping was observed. The release process, which appeared to follow the adherence of platelets to the immune complex, required the action of C6 and perhaps the later-acting components. No evidence could be obtained that a soluble factor was produced which caused the release. Instead, an inhibitor of the release process was detected after incubation of antigen, antibody, and plasma. Antibody preparations capable of giving complement-dependent PCA reactions in rabbits were also shown to induce the release of histamine from platelets in vitro.

Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins).

Purified precursor Hageman factor has been demonstrated to bind to soluble bacterial lipopolysaccharide (LPS, endotoxin) isolated from Escherichia coli 0111:B4, and this complex has been shown to have the capacity to convert prekallikrein to its active form. In addition, LPS-activated Hageman factor substantially reduces clotting times in XII-deficient plasma. The capacity to activate Hageman factor has been demonstrated to reside in the lipid A region of the LPS molecule. Activation of Hageman factor by LPS contrasts with fluid-phase activation (e.g., by kallikrein or trypsin) in that no cleavage to lower molecular weight fragments occurs. High concentrations of LPS inhibit the activity of Hageman factor, probably by a direct LPS-Hageman factor interaction.

Immunological induction of increased vascular permeability. I. A rabbit passive cutaneous anaphylactic reaction requiring complement, platelets, and neutrophils.

Passive cutaneous anaphylaxis (PCA) reactions were produced in rabbits by antibodies to bovine serum albumin. Two types of antibodies were found, each inducing increased vascular permeability, but by means of different mediation systems. One of these antibodies required the presence of complement, platelets, and neutrophils for the induction of the PCA reaction, which was inhibited by antihistamine. This antibody was heat stable, sedimented in the 7S region, and was found in both fast and slow electrophoretic fractions of rabbit gamma-globulin. Homocytotropic antibody was also detected. The PCA reactions induced by this type of antibody did not require platelets or neutrophils and were not inhibited in rabbits depleted of C3 with cobra venom factor. The lesions were, however, prevented by administration of antihistamine.

The first component of the kinin-forming system in human and rabbit plasma. Its relationship to clotting factor XII (Hageman Factor).

The isolation and characterization of the first component of the kinin-forming system in human and rabbit plasma are presented. Functionally, the molecule is the precursor of the activator of prekallikrein (Pre-PKA) and evidence is presented that it is identical with Hageman factor (clotting factor XII). The component from each plasma possessed similar characteristics. This molecule was found to have a mol wt of 110,000 and sedimentation rate of 4.6S. It migrated in electrophoresis as a beta-globulin, having an isoelectric point of 6.1. Upon activation with glass, kaolin, diatomaceous earth, ellagic acid, or trypsin, the activated molecule converted purified prekallikrein (prokininogenase) to the active enzyme. Clot-promoting activity was associated with the capacity to activate prekallikrein through each procedure of isolation. The clot-promoting factor was in precursor form, requiring treatment with kaolin or trypsin to gain activity. Evidence indicated that the protein was Hageman factor (factor XII): it promoted clotting of factor XII-deficient, but not Factor XI- or IX-deficient plasma, and did not convert fibrinogen to fibrin it bound to and was activated by kaolin or other negatively charged particles in the presence of chelating agents; the activation by kaolin could be prevented by pretreating the kaolin with hexadimethrine bromide (H Br); prekallikrein-activating and clot-promoting activities were identical in their physical properties; and the prekallikrein activator could not be detected in Hageman factor-deficient plasma. Activation of Hageman factor was accompanied by cleavage of the molecule into several fragments, one of which possessed prekallikrein-activating (PKA) and clot-promoting properties. The PKA fragment sedimented at 2.6S and by gel filtration was found to have a molecular weight of 32,000. The PKA possessed only 1/50 the clot-promoting capacity of the freshly activated native molecule.

Acute immune complex disease in rabbits. The role of complement and of a leukocyte-dependent release of vasoactive amines from platelets.

By depletion of C3 from rabbits undergoing acute experimental immune complex disease with an anticomplementary factor in cobra venom, it has been possible to demonstrate that deposition of the complexes in arteries and glomeruli does not require the complement components reacting after C2. Immunological reactions, in which platelets release their vasoactive amines, have been examined in rabbits undergoing immune complex disease. A correlation was obtained between the presence of a complement-independent reaction which required blood leukocytes, antigen and platelets, the deposition of immune complexes, and the induction of glomerulonephritis. C3 depletion did, however, have a marked alleviating effect on the severity of the arterial lesions. Neutrophil accumulation and the subsequent necrotizing arteritis were prevented. In contrast, the character and severity of the glomerulonephritis was not altered by depletion of later-acting complement components.

Plasma prekallikrein: isolation, characterization, and mechanism of activation.

The precursor of the kinin-forming enzyme, prekallikrein, was isolated from rabbit plasma protected from activation during preparatory procedures. Prekallikrein was shown to be a 4.5S gamma(1)-glycoprotein with an isoelectric point of 5.9 and a mol wt of 99,900. The proenzyme was activated at neutral pH by an enzyme from rabbit or human plasma we have termed prekallikrein activator (PKA) or by trypsin. Prekallikrein was activated by PKA by a process of enzymatic scission. This resulted in the appearance of two fragments; the larger of these possessed kallikrein activity.

Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils, and a platelet-activating factor.

We have studied the leukocyte-dependent mechanism of histamine release (LDHR) from rabbit platelets, a complement-independent mechanism which has been implicated in the deposition of immune complexes in acute serum sickness of rabbits. It was found by chromatography and passive transfer of serum from immunized rabbits that the antibody responsible for the LDHR was of IgE type. By electron microscope study of the reaction, the leukocyte involved in agglutination of platelets and release of their histamine content was identified as the basophil. Upon addition of antigen, basophils sensitized with IgE degranulated, released their histamine content and a platelet-activating factor (PAF) that caused aggregation of platelets and release of their histamine. Conditions of preparing and preserving PAF activity and some properties of this factor have been elucidated. LDHR must, therefore, be considered as an immediate hypersensitivity-type mechanism which may link allergic reactions with immunologic disease associated with severe structural injury.

Detection of active kallikrein in induced blister fluids of hereditary angioedema patients.

Six suction-induced blister fluids obtained from five patients with hereditary angioedema (HAE) contained active kallikrein, whereas only two blister fluids obtained from eight normal volunteers contained small amounts of this activity. Kallikrein was present in large amounts of HAE blister fluids as assessed by its ability to liberate smooth-muscle-contracting activity from purified high molecular weight kininogen. It was inhibited by purified antibodies specific for plasma prekallikrein and also by purified C1 inhibitor, but not by antibodies specific for C1s. These observations suggest that activation of the Hageman-factor-dependent pathways occurs in the tissues of HAE patients, and once generated, active kallikrein persists in these tissues.

Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum.

A small fragment of C3, called C3a, which has smooth muscle contracting activity, was isolated by three different methods. At pH 8.6, C3a behaved as cation, and using the Archibald method, its mol wt was determined to be 7000. A specific antiserum to C3a showed the fragment to be antigenically distinct from the rest of the C3 molecule, i.e., the C3b portion. The same antiserum and an anti-whole C3 were able to inhibit the biologic activity of C3a. In addition to anaphylatoxin activity, leukocyte chemotactic activity was shown to reside in C3a. Treatment with trypsin caused the cationic fragment to become anionic and abolished the anaphylatoxin but not the chemotactic activity. C3a fragments with identical biologic activity and comparable cationic properties, as determined by acid disc electrophoresis, were obtained by treatment of C3 with C3 convertase, C3 inactivator complex, trypsin, and plasmin. Thrombin produced a similar C3 fragment which was inactive. It was concluded that C3a corresponds to an unusually basic portion of C3 which may be liberated by attack of a variety of enzymes on a highly susceptible region of the native C3 molecule. C3b was cleaved by trypsin and less efficiently by thrombin or plasmin into two antigenically distinct pieces: the larger C3c fragment corresponding to beta(1A) and the smaller C3d fragment to alpha(2D) of aged serum. The c- and the d-fragments were separated and characterized. Isolated C3a rapidly lost its anaphylatoxin activity when treated with small amounts of a partially purified, thermolabile 10S alpha-pseudoglobulin of human serum. The conditions of inactivation suggested an enzymatic reaction. The anaphylatoxin inactivator also destroyed the activity of C5-derived anaphylatoxin and of lysyl bradykinin.

Activation of Hageman factor in solid and fluid phases. A critical role of kallikrein.

The activation of Hageman factor in solid and fluid phase has been analyzed. Activation of highly purified Hageman factor occurred after it interacted with and became bound to a negatively charged surface. Activation was observed in the absence of enzymes that are inhibitable with diisopropylfluorophosphate, phenyl methyl sulfonyl fluoride and epsilon-amino-n-caproic acid. The binding of [(125)I]Hageman factor to the negatively charged surface was markedly inhibited by plasma or purified plasma proteins. Activation of Hageman factor in solution (fluid phase) was obtained with kallikrein, plasmin, and Factor XI (plasma thromboplastin antecedent). Kallikrein was greater than 10 times more active in its ability to activate Hageman factor than plasmin and Factor XI. The data offer a plausible explanation for the finding that highly purified kallikrein promotes clotting of normal plasma. In addition, the combined results of this and previously reported data from this laboratory indicate that the reciprocal activation of Hageman factor by kallikrein in fluid phase is essential for normal rate of activation of the intrinsic-clotting, kinin-forming, and fibrinolytic systems. Activation of Hageman factor was associated with three different structural changes in the molecule: (a) Purified Hageman factor, activated on negatively charged surfaces retained its native mol wt of 80-90,000. Presumably a conformational change accompanied activation. (b) In fluid phase, activation with kallikrein and plasmin did not result in cleavage of large fragments of rabbit Hageman factor, although the activation required hydrolytic capacity of the enzymes. (c) Activation of human Hageman factor with kallikrein or plasmin was associated with cleavage of the molecule to 52,000, 40,000, and 28,000 mol wt fragments. Activation of rabbit Hageman factor with trypsin resulted in cleavage of the molecule into three fragments, each of 30,000 mol wt as noted previously. This major cleavage occurred simultaneously with activation.

Mediation systems in bacterial lipopolysaccharide-induced hypotension and disseminated intravascular coagulation. I. The role of complement.

We have studied the role of complement in lipopolysaccharide (LPS)-induced hypotension and disseminated intravascular coagulation (DIC) by comparing the effects of injection of three preparations of LPS from E. Coli 0111:B4, S. minnesota Re595, and S. marcescens. Injections of nonlethal doses of these LPS preparations into normal rabbits produced decreases in mean arterial blood pressure during a 5-h period. When rabbits treated with cobra venom factor (CoF) to deplete C3 were injected with the various LPS preparations, mean arterial pressures fell at a rate and extent essentially identical to that observed in normal rabbits. Rabbits genetically deficient in C6 also demonstrated LPS-induced hypotensive changes. Only minimal, or no changes in plasma C3 levels or serum CH50 values were detected in normal rabbits after LPS injection. Hypotensive changes were also induced in rabbits when complement was rapidly activated by intravenous injection of CoF. In contrast to the hypotension induced by LPS, the fall in arterial pressure associated with the consumption of complement was short lived and required the rapid consumption of considerable amounts of C3. The occurrence of DIC noted in normal rabbits injected with each preparation of LPS was not inhibited in either rabbits treated with cobra factor or in C6-deficient rabbits. The DIC was most pronounced after injection of Re595 and S. marcescens LPS. Injection of the various LPS preparations produced a rapid disappearance of circulating neutrophils and mononuclear cells, which occurred with the same kinetics and to the same extent in normal, CoF-treated, and C6-deficient rabbits. Injection of either Re595 LPS or S. marcescens LPS produced a biphasic disappearance of circulating 51Cr-platelets. In contrast, injection of 0111:B4 LPS affected only slightly the rate of disappearance of 51Cr-platelets. Depletion of C3 by cobra factor treatment had no effect on the disappearance of platelets in animals injected with 0111:B4. In marked contrast cobra factor treatment greatly reduced the initial rapid disappearance of platelets in rabbits injected with either Re595 or S. marcescens LPS, but had no effect in the secondary disappearance phase.

Surface and fluid phase activities of two forms of activated Hageman factor produced during contact activation of plasma.

The ability of the two forms of activated Hageman factor (HFa) produced during contact activation of plasma to activate prekallikrein and factor XI was studied. alpha-HFa, defined as an 80,000 mol wt two-chain enzyme which remains bound to the surface was capable of cleaving surface-bound prekallikrein and factor XI. beta-HFa, a 28,000 mol wt single chain molecule, released from the surface during contact activation was able to cleave prekallikrein but showed no activity on factor XI. Cleavage of prekallikrein by beta-HFa occurred irrespective of whether the substrate was surface-bound or in solution. Cleavage of factor XI occurred only when it was surface bound and only the alpha-form of HFa was capable of this proteolytic action. Factor XI was found to remain bound to the surface while prekallikrein and kallikrein rapidly dissociated from the surface into the supernate. These findings suggest that the initiation of intrinsic coagulation through the activation factor XI is a localized event occurring at the site of contact activation and is the result of the action of alpha-HFa. By contrast, kinin generation and fibrinolysis resulting from the formation of kallikrein can be initiated either at the site of contact activation, by alpha-HFa action, or throughout the plasma, by beta-HFa; further dissemination of these activities is assured by the rapid dissociation of kallikrein itself from the surface.

Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.