C-reactive

protein selectively

generation

of reactive

monocytes

and neutrophils

Janice

M. Zeller*,I

Departments

of

and

*Medical

enhances

the intracellular

products

by lgG-stimulated

oxygen Brenda

Nursing

L. Sullivan1

and

Tlmmunology/Microbiology,

C-reactive

protein

Rush-Presbyterian-St.

Luke’s

Medical

Center,

Chicago,

Illinois

Abstract:

The

(CRP), when munoglobulin

acute

phase

heat-aggregated G (IgG)

Fc

protein, (Agg-CRP), receptor-mediated

potentiates

(CRP),

imluminol-

enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of mono:y tes and neutrophils to Agg-CRP, superoxide anion elease was measured by cytochrome c reduction. In addiion, the extracellular release of hydrogen peroxide was istinguished from hydrogen peroxide generation using a henol red oxidation assay. Finally, a flow cytometric deermination of dichiorofluorescin (DCFH) oxidation was mployed as an index of intracellular peroxide producion. Although Agg-CRP alone did not stimulate hydroen peroxide generation by either monocytes or neurophils, it significantly enhanced hydrogen peroxide eneration in response to heat-aggregated IgG (Agg-IgG). n contrast, Agg-CRP did not enhance the extracellular lease of either hydrogen peroxide or superoxide anion rom Agg-IgG-stimulated cells. The capacity of Agg-CRP 0 enhance selectively intracellular oxidative product eneration was confirmed when measuring DCFH oxidaion in Agg-IgG-stimulated cells. To evaluate whether his selective enhancement of intracellular oxidative ents could be attributed, at least in part, to a scavengng effect of Agg-CRP, a cell-free oxygen radical-generatng system was employed. Agg-CRP did not significantly iminish the lucigenin-amplified CL response induced by he xanthine/xanthine oxidase icate that although Agg-CRP eneration of reactive oxygen nd neutrophils, extracellular

reaction. These results enhances the intracellular intermediates by monocytes release of those products

in-

C,,

eroxide

Words: C-reactive protein . chemiluminescence . p/zagocytes . superoxide anion

.

hydrogen

NTRODUCTION uring is,

hepatic

arkedly

episodes synthesis

enhanced

of infection, and

[1].

inflammation, or tissue necroof acute-phase proteins are Plasma bevels of C-reactive protein release

acute-phase

protein

in

humans,

in-

acute-phase protein

response, human

and

interactions phagocytic

CRP comprises five noncovalently associated subunits at-ranged in cyclic symmetry [5]. Certain laboratories have reported that native pentameric CRP can bind to human leukocytes [6, 7], whereas others indicate that optimal cell binding of CRP is dependent on prior aggregation or complex formation with a ligand [8-11]. It is recognized that engagement of membrane binding sites by CRP can influence cell function, as CRP deposition on particle surfaces facibitates phagocytosis by monocytes [12] and neutrophils [13]. Whereas CRP has the capacity to promote phagocytosis, it apparently lacks the ability to stimulate directly a respiratory burst response [10, 11, 14]. Our laboratory reported, based on data obtained utilizing luminol-enhanced chemiluminescence (CL) measurements, that aggregated forms of CRP markedly potentiate respiratory burst stimulation of human monocytes [11] and neutrophils [10] following immunoglobubin G (IgG) Fc receptor ligation. The light emitted by phagocytic cells during a luminol-CL response has been attributed to the generation of reactive oxygen intermediates in the presence of myeloperoxidase [15]. Although luminol-CL is a sensitive measure of respiratory

sites

ot influenced by cell interaction with Agg-CRP. It is empting to speculate that CRP can selectively boost the icrobicidal activities of monocytes and neutrophils wihin an inflammatory site by amplifying the intracellular eneration of reactive oxygen products without increasng damage to surrounding normal tissues. J. Leukoc. iol. 52: 449-455; 1992.

prototypic

the role for CRP in the between this acute-phase cells have been explored.

those

is

the

crease as much as 1000-fold within 24 h of the onset of acute inflammation [1]. Although a protective robe for CRP in the acute-phase response has not been completely delineated, it has been observed that CRP confers resistance against lethal bacterial infections [2, 3] and tumor growth [4] in experimental animals. To gain more information concerning

purpose

burst

activity

oxidative

of

in

elaboration of

phagocytic

products

the

detected

remain present

study

cells,

the

by

luminol-CL

incompletely was

nature and

understood.

to better

CRP potentiation of IgG-induced responses. The capacities of phagocytes ide anion (O2) and hydrogen pet-oxide

exact

characterize

of their

The the

respiratory burst to generate superox(H2O2) were meas-

Abbreviations: ANOVA, analysis of variance; CRP, C-reactive protein; Agg-CRP, heat-aggregated CRP; CL, chemiluminescence; DCFH, dichlorofluorescin; Agg-IgG, heat-aggregated IgG; DCFH-DA, dichlorofluorescin diacetate; IgG, immunoglobulin G; MN, mononuclear leukocyte; NaOH, sodium hydroxide; OD, optical density. Reprint requests: Dr. Janice M. Zeller, Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Received February 20, 1992; accepted May 26, 1992. This work was presented, in part, to the Society for Leukocyte Biology. Zeller, J.M., Frank, B.L., Dickinson, R. (1987) Superoxide and peroxide production by human polymorphonuclear leukocytes (PMNL) is enhanced by aggregated C-reactive protein (Agg-CRP). j Leukoc. Biol. 42, 343.

Journal

of Leukocyte

Biology

Volume

52,

October

i992

449

ut-ed with ferricytochrome c reduction [16, 17] and phenol red oxidation [17, 18] assays, respectively. To evaluate selectively intracellular pet-oxide production, dichlorofluorescin oxidation was measured [19, 20]. Results of these studies indicated that aggregated CRP mat-kedly potentiated the respiratory burst response of IgG-stimulated monocytes and neutrophils as reflected in the assays measuring intracellular oxidative

product

peroxide

anion

generation. and

In

hydrogen

contrast,

the

peroxide

by

release

of

su-

IgG-stimulated

phagocytic cells was only minimally altered by CRP. These data suggest that during an acute-phase response, CRP may regulate phagocytic cell responses and thereby influence host defense by selectively enhancing intracellular oxidative events without concomitantly amplifying oxygen radical release.

MATERIALS

AND

METHODS

Materials Sepharose 4B and Sepharose S-200 were put-chased from Pharmacia Fine Chemicals (Piscataway, NJ). DE-52 was obtamed from Whatman (Clifton, NJ). 2’,7’-Dichlorofluorescm diacetate (DCFH-DA) was purchased from Eastman Kodak (Rochester, NY). Ferricytochrome c (horse heart), cytochalasin B, horseradish peroxidase (type II), phenol red, lucigenin, xanthine, xanthine oxidase (grade IV, milk), and superoxide dismutase were obtained from Sigma Chemical Co. (St. Louis, MO). Human IgG was purchased from Cutter Laboratories (Berkeley, CA). Plastic flasks and microtiter plates were obtained from Flow Laboratories (McLean, VA). All other chemicals were of reagent grade quality and put-chased from local vendors.

Isolation

and

purification

of CAP

Cell preparation Peripheral venous blood samples were collected from healthy human volunteers and transferred to heparinized tubes. Mononuclear leukocytes (MNs) were separated from neutrophils by density gradient centrifugation [11]. Neutrophils were cleared of contaminating erythrocytes by dextran followed of approximately whereas the

by hypotonic lysis. 30% monocytes neutrophil fraction

The

MN fraction and 70% lymcontained fewer

450

staining [23]. as determined

Journal

of Leukocyte

Cell by

viability trypan

Biology

Volume

52,

October

1992

release

was

established were added salt

measured

using

protocols [17, to microtiter

24].

an

adaptatior

Briefly, MNs plates in Hanks

0]

containing 100 M ferricytochrome B at pH 7.4. Release was initiatec with stimulus addition and was allowed to proceed at 37#{176}C Samples were read in a Titertek multiscan plate reader (Flos Laboratories) at 15-mm intervals for 60 mm. The differenc in absorbance at a 550-nm wavelength in wells containing o lacking 40 g/ml superoxide dismutase (zOD550) wa proportional to the amount of O2 produced. The change itoptical density at 550 nm (OD550) was converted to nano moles of O2 released using the extinction coefficient E550 = 21 x 10 M1 cm [25]. Superoxide anion release, as evaluated with this detection system, was linear up to 60 mm aftei stimulus addition. The rate of O2 release from Agg. IgG-stimulated cells was comparable to that previously ob served for human monocytes and neutrophils following IgC Fc receptor cross-linking [26-28]. and

5 g/ml

Phenol

solution

cytochalasin

red oxidation

Hydrogen

pet-oxide

generation

was

measured

by

quantifyin

the horseradish peroxidase-catalyzed oxidation of pheno red [17, 18]. Purified monocytes or neutrophils (1.25 x 10 cells) were added to microtiter plates containing phosphate buffered saline with 0.2 g/L phenol red, 19 U/mb horseradisF peroxidase, and 5 jg/ml cytochalasin B at pH 7.0. Followin stimulus addition and incubation at 37#{176}Cfor up to 60 minj the reaction was terminated with 1 M sodium hydroxid ( NaOH). Absorbance was measured at 600 nm. Hydroger peroxide production was quantified by reference to a stan dard curve generated at each experiment. To measure selectively H202 release from cells, microtitei plates were centrifuged after cell incubation with stimulus, were

supernatants

treated

with

were

transferred

NaOH

and

Dichlorofluorescin

to

H2O2

fresh

was

plates.

quantified

Sample

as above

oxidation

Purified monocytes or neutrophils were incubated with 5 i1 DCFH-DA for 15 mm at 37#{176}Cto allow for product boadin and deacetylation as previously described [19, 20]. Followin stimulus addition to 0.2 x 106 cells, samples were incubatec at 37#{176}C for 60 mm and immediately analyzed using Coulter Epics C flow cytometer (Coulter Electronics, Hialeah, FL) equipped with a 4-watt argon laser run at 30( mW with a 75 mV high-voltage setting and an excitatior wavelength of 488 nm. All fluorescence was measured usin logarithmic amplification. Cells were discriminated on th basis of forward angle bight scatter and bog 90#{176} light scattei parameters. For each histogram, 5000 cells were analyzed. Data were reported as the mean of the fluorescence intensit) channels,

Lucigenin

was routinely greater blue dye exclusion.

c reduction

anion

balanced

(256 units).

than 2 % contaminating monocytes and lymphocytes. In certam experiments, monocytes were separated from lymphocytes by attaching MNs to fibronectin-gelatin-coated flasks, removing nonadherent cells, and detaching monocytes with 5 mM ethylenediaminetetraacetate [22]. These preparations contained more than 96% monocytes as determined by peroxidase than 98%

Superoxide of previously neutrophils

and

CRP was isolated from pooled human fluids by sequential chromatography on phosphocholine coupled to Sepharose 4B, DE-52, and S-200 columns as previously described [21]. Preparations were free of antigenically detectable IgG, 1gM, serum amyboid P component, lipoproteins, and complement components Clq, C4, and C3. On characterization by sodium dodecyl sulfate electrophoresis, all preparations showed a single band of 24,000 daltons. CRP was heat aggregated (Agg-CRP) prior to use [11] and subsequently added to cells in the presence and absence of heat-aggregated IgG (Agg-IgG), prepared as previously described [11].

sedimentation consisted phocytes,

Ferricytochrome

a

three-decade

log

scale

in

arbitrary

lo

chemiluminescence

Xanthine (0.2 mM) glass liquid scintillation

and

lucigenin vials in

(0.2 either

mM) were the presence

added or

tc ab

sence of 100 g/ml Agg-CRP. Samples were allowed to dar adapt for 30 mm. Oxygen radical generation was elicitec with the addition of 0.1 U/ml xanthine oxidase. Light emis sion was detected at regular intervals for at least 30 mm us ing a 6895 Beta Trac liquid scintillation counter (TM Ana lytic, Elk Grove Village, IL) set in the out-of-coincidencc mode. Data were reported as counts per minute (cpm).

Statistical Data ing

4

significance

were

analyzed

A

statistically significant t-test. A P value .05 was In certain experiments, values analysis of variance (ANOVA),

Student’s

significant. using an differences test.

for

between

groups

determined

by

differences considered were

usto be

3.

compared

with

significant

a post

hoc

Tukey’s

2

RESULTS

U, U,

Effect

of aggregated

CAP

on ferricytochrome

c reduction

C.) C

The influence anion release

ofAgg-CRP was examined

tion assay. To prevent enhance detection of 5

jzg/ml

Agg-CRP amounts trophils.

on Agg-IgG-induced using a ferricytochrome

engulfment 02 release Table

of Agg-IgG [26], cells

cytochalasin

B.

alone did of superoxide A comparable

not induce anion from concentration

1

c reduc-

[29] were

illustrates

0

superoxide and exposed

that

100

the release of either monocytes of Agg-IgG,

0

thus

0

to

30

60

30

60

U,

0

g/ml

0

substantial or neuhowever,

U,

a. C U,

stimulated the release of 3.4 and 5.1 nmol of superoxide anion from 106 monocytes and neutrophils, respectively. In contrast to our earlier results with luminol-CL [10, 11], AggCRP did not significantly potentiate the reduction of ferricytochrome c by Agg-IgG-stimulated cells. The data in Table 1 were collected 60 mm after stimulus addition. Values obtained at earlier time points (15 and 30 mm) similarly failed to demonstrate significant differences between control and Agg-CRP-treated cells (data not shown). To determine whether the inability of Agg-CRP to augment significantly Agg-IgG-induced O2 release was due to an enhancing effect of cytochabasin B [26, 30, 31], additional experiments were carried out in the absence of the drug. Elimination of cytochalasin B from the assay mixture resulted in a 25% reduction in 02 release from AggIgG-stimulated monocytes and neutrophils. As seen in cytochalasin B-treated cells (Table 1), Agg-CRP failed to potentiate Agg-IgG-induced O2 release (data not shown).

a) 0 >‘

I (I, 4, 0 C

TIme

(minutes)

Fig.

1. Enhancing effect ofAgg-CRP on Agg-IgG-induced hydrogen peroxproduction by human monocytes and neutrophils. Hydrogen peroxide ( H202) production by monocytes (A) or neutrophils (B) was measured in the presence of0.2 g/L phenol red, 19 U/ml horseradish peroxidase, and 5 g/ml cytochalasin B. Cells were incubated for up to 60 mm with either saline (U), 100 tg/ml heat-aggregated CRP (Agg-CRP) (A), 100 g/ml (monocytes) or

ide

Eftect The says

of aggregated

data taken

TABLE

obtained together

1.

CAP on phenol from with

Effect from

the ferricytochrome our earlier data

of Aggregated Human

CRP

Monocytes

on

red oxidation for

c reduction luminol-CL

Superoxide

and

Anion

assug-

200 g/ml (neutrophils) heat-aggregated IgO (Agg-IgG) (#{149}), or 100 ig/ml Agg-CRP with 100 sg/ml (monocytes) or with 200 g/ml (neutrophils) AggIgG (0). The reaction was terminated with the addition of NaOH and absorbance at 600 nm was compared to a reference standard. Data are cxpressed as nanomoles of H202 produced per 106 cells (mean ± SEM of three to five experiments). The values obtained with Agg-CRP alone (A) are not shown in (B) as they overlapped completely with results for saline-

Release

Neutrophils

Ferricytochrome

c reductiona

treated Stimulus

Monocytes

Saline AggCRPb Agg-IgG’

Agg-IgG(l00g/m1)

0 0.36 2.62 3.11 3.36

Agg-CRP

3.36

+

+

.

(25

sg/m1)

Agg-CRP

Monocytes

or

neutrophils

were

added

0.14 0.16 4.32 4.36 5.12 5.12

0.12 ± 0.60 ± 0.64 ± 0.52 ± 0.44 ±

to

microtiter

plates

± 0.i2 ± 0.08 ± 0.72 ± 1.80 ± 0.84 ± 0.84

in

Hanks’

balanced salt solution containing 100 &M ferricytochrome c and 5 g/ml cytochalasin B. Superoxide anion release was measured 60 mm after the addition of the indicated stimuli. The differences in absorbance at 550 nm messured in wells containing or lacking superoxide dismutase (40 sg/ml), were used to calculate the amount of superoxide anion generated. Data are cxpressed as nanomoles of ferricytochrome c reduced per 106 cells (mean ± SEM of five experiments). bFinal concentration of aggregated-CRP (Agg-CRP) in all reaction mix-

tures

was

‘Agg-IgG,

100 .tg/ml. heat-aggregated

neutrophils.

Neutrophils

IgG.

gest that while Agg-CRP potentiates the generation of reactive oxygen products by Agg-IgG-stimulated cells, it fails to increase extracelbular levels of these reactive oxygen species. For confirmation of these observations, additional experiments were undertaken to quantify H202 generation by and its release from phagocytic cells. Hydrogen peroxide generation (Fig. 1) was measured by incubating either monocytes

(panel presence

A)

or neutrophils (panel and absence ofAgg-CRP

in the at 37#{176}C

and terminating the reaction with NaOH. Phenol red oxidation, measured in this way, reflects contributions of both intraceblular and extraceblular H202 compartments. Agg-C RP alone did not induce H2O2 production above that spontaneously generated by saline-treated control cells. In contrast, Agg-IgG stimulated additional H2O2 production in a timedependent

Zeller

B) with Agg-IgG for up to 60 mm

and Sullivan

manner.

CRP

When

stimulation

Agg-CRP

was

of reactive

oxygen

added

in combina-

products

451

tion with (P < .05; responses

Agg-IgG, significant potentiations of monocyte P < .01) and neutrophil (P < .001; P < .05) were observed at the 30- and 60-mm time points,

respectively. To evaluate duced H202 were incubated Agg-CRP minated wells, data

the influence of Agg-CRP release from monocytes and with Agg-IgG in the presence

for up to 60 mm by pelbeting cells,

at 37#{176}Cand the reaction transferring supernatants

was terto fresh

supernatants with NaOH. The mm after stimulus addition, illustrate that monocytes neutrophils release negligible bevels of H2O2 in response to Agg-CRP. The amount of H2O2 released following stimulation with Agg-IgG was approximately 50% ofthat measured in the total H202 generation assay at comparable stimulus concentrations. In contrast to data obtained when H2O2 bevels were measured in cells

and treating cell-free in Table 2, collected

on Agg-IgG-inneutrophils, cells and absence of

and

culture

60 and

supernatants

taken

together,

Agg-CRP

U

C U U, 1

0

0

20

40

60

Agg-IgG

U-

80

120

100

(iglmi)

did

not significantly potentiate the release of H2O2 from AggIgG-treated cells. Similar results were obtained when H2O2 release was quantified 30 mm after stimulus addition or in the absence of cytochalasin B (data not shown). These observations confirm that while Agg-CRP can enhance the generation of reactive oxygen products by phagocytic cells, the extracellular level of these products is not increased by exposure to Agg-CRP.

C C U, -C

U

140

C U,

5

120

100

Effect of aggregated oxidation

CAP

on dichlorofluorescin 80

The observation that Agg-CRP could influence Agg-IgG-induced generation of H2O2 by phagocytic cells without altering the extracellubar levels ofeither O2 or H2O2 provides indirect evidence for the ability of Agg-CRP to modulate selectively intracellular oxidative events. To evaluate intracellular production of oxygen metabobites more directly,

60

40 0

dichboroflourescin oxidation was measured (Fig. 2). Monocytes (panel A) or neutrophils (panel B) were incubated with DCFH-DA for 15 mm and exposed to varying concentrations of Agg-IgG in the presence and absence of Agg-CRP for 60 mm at 37#{176}C. Mean channel fluorescence data obtamed from both monocytes and neutrophils revealed that Agg-CRP alone induced only small increases in intracellular peroxide production (Fig. 2; y axis). Larger dose-dependent

TABLE

2.

Effects from

of Aggregated CRP Human Monocytes

on Hydrogen and Neutrophils Phenol

Stimulus Saline Agg-CRP5 Agg-IgG’ Agg-IgG

Peroxide

+

Agg-CRP

Fig.

Journal

of Leukocyte

0.19 0.22 1.06

± 0.12 ± 0.08 ± 0.20

1.32

±

0.28

1.58

±

0.31

to microtiter plates in phosphatered, 19 U/ml horseradish peroxirelease was measured for 60 mm The absorbance ofsodium hydroxat 600 nm and H202 content was

52,

CRP-induced

October

in DCFH oxidation as a stimulus. The

preparations exposed DCFH oxidation in

Neutrophils

Volume

Aggregated

increases was used

± 0.02 ± 0.08 ± 0.13

Biology

2.

enhancement

80

100

(sg/mi) of

intracellular

peroxide

production by Agg-IgG-stimulated monocytes and neutrophils. Human monocytes (A) and neutrophils (B) were incubated with 5 sM 2’,7’-dichlorofluorescin diacetate for 15 mm at 37#{176}C.Cells were stimulated with the indicated doses of heat-aggregated IgG (Agg-IgG) in the presence of 0 (#{149}), 50 (0), or 100 (U) sg/ml heat-aggregated CRP. After a 60-mm incubation at 37#{176}C,samples were analyzed using an Epics C flow cytometer. Data are expressed as mean channel fluorescence (mean ± SEM of seven experiments).

quantified by reference to a standard curve. Data are expressed as nanomoles of H202 released per 106 cells (mean ± SEM of five experiments). 5Final concentration of aggregated CRP (Agg-CRP) in all reaction mixtures was iOO g/ml. ‘Monocytes were stimulated with 100 ig/ml aggregated IgG (Agg-IgG), whereas neutrophils were stimulated with 200 zg/ml Agg-IgG, in order to generate comparable amounts of hydrogen peroxide.

452

6&

Release

0.03 0.19 1.03

. Monocytes or neutrophils were added buffered saline containing 0.2 g/L phenol dase, and 5 g/ml cytochalasin B. H202 after the addition ofthe indicated stimuli. ide-treated cell supernatants was measured

40

Agg-IgG

red oxidationS

Monocytes

20

1992

were observed addition of

to Agg-IgG a dose-dependent

when Agg-CRP

markedly manner.

Agg-IgG to cell potentiated Statistical

significance of these changes was calculated with ANOVA and a post hoc Tukey’s test. For monocytes, the potentiating effect was statistically significant (P < .05) at all doses of Agg-IgG when 100 g/m1 Agg-CRP was employed but only at the 10 and 20 jg/ml Agg-IgG doses when 50 sg/ml AggCRP was employed. Similarly, statistical significance (P < .05) was achieved in neutrophils with 100 jsg/ml AggCRP at 10, 20 and 50 ig/ml Agg-IgG; however, 50 sg/ml Agg-CRP significantly potentiated the respiratory burst response only to the bower doses (10 and 20 g/ml) of AggIgG. At all concentrations of Agg-IgG and Agg-CRP tested, monocytes and neutrophils responded with increases in fluorescence intensity as uniform cell populations. These DCFH oxidation data indicate that while Agg-CRP alone largely fails to stimulate the production of intracellular peroxide, it significantly enhances intracellular peroxide

by

production

Agg-IgG-stimulated

monocytes

and

neu-

potentiated

trophils.

Effect

of aggregated

oxidase-induced

CAP

on xanthine/xanthine

lucigenin

chemiluminescence

To determine whether the selective lar oxidative events by Agg-CRP attributed to a scavenging effect idants, the effect of Agg-CRP

potentiation

of

intracellu-

could, at beast in part, of released extracellular on xanthine/xanthine

be oxoxi-

the CL response CRP (106 M)

by 94%,

the

not

significantly

did

(data not shown). These selective enhancement of Agg-CRP does not involve reactive oxygen products.

presence

of

100

reduce

tg/ml

of

phagocyte

CL

data indicate that the apparent intracellular oxidative events the extracellular scavenging

respiratory

stimubators,

nonphagocytosable mediates generated bacterial killing [33] tribute to tissue injury The respiratory but-st tron transport system to molecular oxygen ofa stimulus-induced dase is activated, in components including bly,

a 45-kd

flavoprotein

by of

response is triggered by or cell attachment to surfaces [32]. Reactive oxygen interwithin a phagobysosome are critical for but, when released from the cell, conwithin an inflammatory site [34, 35]. enzyme, NADPH oxidase, is an electhat transfers electrons from NADPH to generate #{176}2, the immediate product oxidative burst [32, 36]. NADPH oxipart, by the association of membrane cytochrome b558 [37-39] and, possi40],

with

at

least

two

burst tiated [10,

in human forms

phagocytic cells. We observed of CRP alone failed to induce

as measured by luminol-CL, IgG-Fc receptor-mediated 11].

In

contrast,

aggregated

aggregated respiratory CRP

failed

but-st

other We

than luminol-CL observed that

to assess respiratory while Agg-CRP

present data red oxidation with luminol-CL

oxidation

cytochrome that

and

were made, when cell c reduction

Agg-CRP

has

the generation of following monocyte

the

ca-

oxidative or neu-

obtained with DCFH and whole cell measurements confirmed our earlier [10, 11], supporting the hypothesis

to

species

for

over Most

required

for

20 years [47], studies indicate buminol-CL

is still that include

indicate

that

such

as

catalase

unpublished

preboad

cells

when

Agg-IgG

alone

is

used

as

a

is largely reflective of intracellular oxfailed to observe a significant reduction the addition of large cell-impermeant or

superoxide

observations). with

dismutase

In addition,

luminol,

wash,

and

we still

G.M.

were

detect

able Agg-

IgG-induced CL G.M. Zebler, unpublished observations). Other agents such as phorbol esters [51], certain unopsonized bacteria [52], and latex particles [53] also appear to elicit preferentially an intracellular oxidative burst response. Two additional assay systems employed in the present studies, ferricytochrome c reduction and phenol red oxidation, utilize barge impermeant molecules as detection markers; thus they selectively measure the release of O2 and H2O2, respectively, from stimulated cells. The phenol red oxidation assay can be adapted to measure whole cell H2O2 production by lysing cells with NaOH and performing measurements in combined cell lysates and supernatants [17, 24]. DCFH oxidation was initially utilized to measure intracellular peroxide production in neutrophils by Bass and coworkers [19]. Recently our laboratory adapted this method for use with human monocytes [20]. While assays to measure H202 or O2 release from cells are employed widely to characterize respiratory burst activation, they share the limitation

of

failing

to

measure

intracellular

oxidative

events.

There tamed

is not always a strong cot-relation between results obin luminol-CL and either #{176}2 or H2O2 release assays [46]. This may be related to the pools of oxidative product measured within each assay or alternatively to the nature of oxidative species detected. The observation that Agg-CRP increases intracellular ac-

ag-

cumulation of reactive oxygen their release from Agg-IgG-stimulated trophils supports earlier evidence traceblular versus extraceblular tially regulated [42-45]. Our Agg-CRP enhances phagocyte through FcyRII but not FcyRI

potentiate

has

been

(Fc-yRI,

burst actisignificantly

Zeller

burst activation defined process.

molecular

Zebber,

phagocyte respiratory but-st activation with serum-opsonized zymosan or phorbol myristate acetate [10, 11]. The put-pose of the present study was to further characterize the effect of Agg-CRP on the Agg-IgG-mediated response by utilizing assays vation.

The phenol results

scavengers

CRP potenactivation

to

DCFH

or when suggest

amplify selectively at intracellular sites activation with Agg-IgG.

stimulus, luminol-CL idative events, as we in luminol-CL with

cytosolic

that while a respiratory

to

laboratory

components, p47[phox] and p67[phoxj [411. In resting neutrophils, the major proportion of cytochrome b558 is localized within the specific granule fraction [37-39] with lesser amounts recovered from plasma membranes [37-39]. Previous studies have documented that neutrophils selectively generate reactive oxygen products within either intracellular or extraceblular compartments [42, 43], dependent on the chosen stimulus [44, 45], receptor availability [43], ionic composition of the bathing buffer [43], and activation state ofthe cell [42]. The origins ofintracellular versus extracellubar oxidative product generation have not been identified; however, studies with neutrophil cytoplasts have linked these events with activation of plasma membrane and specific granule oxidase pools, respectively [45]. Although lacking in specific granules, there is evidence that mononuclear phagocytes generate oxidative products both intracellularly and extracellularly [46]. Our laboratory had previously reported that CRP, an acute-phase reactant, could influence respiratory burst activation gregated

when

H2O2, myeboperoxidase, and a halide [15]. There is evidence that luminol can penetrate the cell [48] and thus can theoretically measure the intracellular as well as extracellubar elaboration of reactive oxygen intermediates. Others have reported that the proportion of a luminol-CL response that is reflective of intracellular events is related to buffer constituents [49] and pH [50], stimulus type [51] and concentration [43], and state of cell activation [42]. Results from our

uptake,

[37,

data

pacity products trophil

the

burst

particle

employed

These

ate respiratory an incompletely

DISCUSSION

soluble

were

measured.

burst

red oxidation measurements influence phenol red oxidation

that luminol-CL is, at least in part, a measure of intracellubar oxidative events. Luminob-CL, although utilized to evalu-

Agg-

lucigenin

oxidative

phenol did not

supernatants was

dase-induced lucigenin CL was measured. The addition xanthine oxidase to xanthine in the presence of bucigenin resulted in light emission that could be maximally detected after 12 mm. Whereas 10 M superoxide dismutase reduced

The

the

whole cell Agg-CRP

documented FcyRII,

burst

response

intracellular

and

respiratory

tions

and

Sullivan

to

that Fc-yRIII)

CRP

stimulation

species

but does not enhance monocytes and neuthat the generation of inoxidative products is differenlaboratory has reported that respiratory burst activation or FcyRIII [54]. Although it all three human IgG Fc receptors are capable of triggering a [55], their respective contribu-

extracellular

of reactive

events

oxygen

have

products

not

been

453

appreciated. Fc’yRII quit-ed products Agg-CRP in

There

is

evidence

that

ligation

results in calcium mobilization for intracellular generation [43]. It would be of interest enhances Agg-IgG-induced

human Previous

monocytes studies

and neutrophils. in our laboratory

of

monocyte

[56, 57], an event reof reactive oxygen to determine whether calcium mobilization and

others

revealed

bind to IgG-containing involvement of both and fibronectin-mediated

immune complexes cell- and IgG-binding potentiation of

[62, 63], but the sites in the CRPrespiratory burst

activation remains to be determined. In summary, the present study demonstrated CRP enhanced the detection of reactive oxygen ates by Agg-IgG-stimulated phagocytes when

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The authors gratefully acknowledge the expert technical assistance of Janice Caliendo and Kathleen Holevinsky and the excellent secretarial support of Mary Rolfe-Shaw. We also thank Dr. Claes Dahlgren for helpful discussions and Dr. Barbara Swanson for statistical consultation. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, AI23030, to J.M.Z.

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