Superoxide generation leukocyte in response Michael J. Thomas Andra S. Willard,t Departments
of Biochemisty*
by the human to latex beads
Catherine and Pamela
C. Hedrick7
and
The
Pathology,
Wake
posure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c. J. Leukoc. Biol. Si: 591-596; 1992. Words:
hydrogen
peroxide
.
phagocytosis
respiratory
burst
INTRODUCTION The formation of O2 by polymorphonuclear leukocytes (PMNs) was first reported by Babion et al. [1] after treating PMNs with latex beads and assessing O2 secretion by the reduction of fernicytochnome c. Studies have repeatedly confirmed [17] the original observation, and O2 has been identified using spin-trapping techniques [4, 13]. Prior to the detection of O2, only H202 formation [3] had been linked to the increased 02 consumption [18, 31] by activated PMNs. Several studies have shown that a variety of actions are stimulated when PMNs are treated with 1.2 to 0.8 jm diameten hydrophobic beads that have been coated with serum or with immunoglobulin G (IgG). Large increases in the rates of glucose oxidation, 02 consumption, H2O2 fonmation, and O2 release have been reported [1, 3, 8, 9, ii, 14, 15, 18, 27, 31, 32]. Untreated hydrophobic latex beads are reported to elicit most ofthe responses obtained with opsonization [2, 9]. However, the question of whether hydrophobic beads cause O2 secretion is disputed. Some laboratories report superoxide dismutase (SOD)-inhibitable fernicytochrome c reduction [1, 2, 30], whereas others were unable to detect significant amounts of O2 [9]. We have used latex beads as a vehicle to study the oxidant chemistry of the phagolysosome [29]. The latex beads carry probe molecules, generally linoleyl alcohol, into the phagolysosome to detect and quantitate oxidants like HO 102, and HOC1. Because O2 generation is not reported after treatment with certain types of latex beads, it was necessary to establish the response modified (CM) latex bead course of these investigations
of the PMN used in these we examined
to
Sharon
Smith
Jian
Pang7
W. Gray
Jerome,t
S. Shirley*
Abstract: In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxymodified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 mm after the initial exposure to beads. However, an 02-producing, NADPH-dependent oxidase is active iS mm after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after cx-
Key
polymorphonuclear
the carboxystudies. In the the response to
Forest
University
Medical
Center,
Winston-Salem,
North
Carolina
CM latex beads coated with linoleyl alcohol by measuring 02 and H2O2 secretion, 02 consumption, NADPHdependent oxidase activity, and the release of lactofennin and myeloperoxidase from granules. We found a significant lag between the initial treatment with beads and the detection of O2, but the other measures process. Our studies imply same physiologic responses cytic
indicated a normal phagocytic that latex particles induce the that characterize other phago-
particles.
MATERIALS
AND
METHODS
General Polystyrene canboxylate-modified hydrophobic latexes (0.732 and 0.822 ,zm in diameter) were obtained from Interfacial Dynamics Corporation. Polyvinyltoluene latex beads (Dow, 2.02 m in diameter) were obtained from Duke Scientific Co. Dulbeccds phosphate-buffered saline (PBS) was from Grand Island Biological Co. Dextran T 500 was from Pharmacia. Fernicytochrome c, phonbol mynistate acetate (PMA), cytochalasin B, NADH, sodium pyruvate, micrococcus lysodeikticus,’ human IgG, and SOD were from Sigma. Linoleyl alcohol was from NuChek Prep., Inc.
Assay
Procedures
PMNs were isolated as previously described [29]. This method yields a preparation that contains greater than 87% granulocytes (generally greater than 95%) with a viability greater than 95% as judged by trypan blue exclusion. Linoleyl alcohol-coated latex beads were prepared as described [29]. IgG-coated beads were prepared by allowing the latex beads to stand overnight at 4#{176}C with 30 mg/mI IgG and then washing the beads five times with deionized water. Oxygen uptake was monitored with a YSI model 53 oximeter using a Clark electrode. A YSI model 25 oxidase meter equipped with a YSI model 2510 oxidase probe was used to measure H2O2 secretion [28]. Hexose monophosphate shunt activity was assessed using the procedure of Long et al. [20]. Degranulation was assessed by the percentage of total lysozyme released into the medium [24, 25]. Cell viability was determined by measuring the percentage of total lactate dehydrogenase (LDH) released by the PMNs [6, 25]. Duplicate
Abbreviations: Gey’s buffered hydrogenase; tate; PMN,
CM, carboxy-modified; DAB, diaminobenzidine; GBSS, salt solution; IgG, immunoglobulin G; LDH, lactate dePBS, phosphate-buffered saline; PMA, phorbol myristate acepolymorphonudear leukocyte; SOD, superoxide dismutase.
Reprint requests: Michael J. Thomas, Department Wake Forest University Medical Center, Medical Winston-Salem, NC 27157-1016. Received September 17, 1991; accepted December
Journal
of Leukocyte
Biology
Volume
of Biochemistry, The Center Boulevard, 13,
51, June
1991.
1992
591
determinations lysozyme and Superoxide SOD-inhibitable
were obtained LDH. production was reduction
at
each
time
point
for
determined by measuring of fernicytochrome
both the [1].
c
Specifically, tubes were prepared on ice containing 1.7 x i06 PMN/ml, 433 ig/ml fennicytochnome c, and 1.1 mM glucose in PBS with a total volume of 3 ml. Half of the tubes also contained 10 g/ml SOD. Control tubes were kept on ice during the experiment. The remaining tubes were incubated for 5 mm at 37#{176}Cand then treated with 100 tl of bead solution. Samples (duplicate or triplicate) were removed at specific intervals, chilled on ice, and then either filtered through 0.45-tim filters or centrifuged at 15,000g before measuring the optical absonptions at 550 and 556 nm. When cytochalasin for 5 mm 1.7
x
B was with 12.5 PMN/ml
106
used, tg/ml (4.2
5 x 106 PMNs were preincubated cytochalasin B and then diluted g/ml cytochalasin B) for the
to cx-
peniment. The activity of the NADP-dependent oxidase was measured in particulate fractions by the method of McPhail and Snyderman [22] without cytochalasin B. Solutions were prepared on ice containing 1.5 x 108 PMNs in 3 ml PBS and incubated for a total of 20 mm. The following treatments were evaluated: (1) incubation on ice with the addition of 50 l linoleyl alcohol-coated CM latex beads (5 x i0 beads/ml) 5 mm after the start of the experiment; (2) incubation at 37#{176}Cfor 5 mm followed by treatment with 50 l of beads; (3) incubation at 37#{176}Cwithout beads; and (4) incubation for 5 mm at 37#{176}Cfollowed by treatment with 1 l PMA (33 ng/ml final concentration). All samples were treated with 30 jl of sodium azide 4 mm after the start of the experiment. At the end of the incubation period the tubes were treated with 15 ml ice-cold PBS and placed on ice. PMNs were pelleted at l8Og (4#{176}C).The pellet was resuspended in 0.34 M sucrose to a concentration of 2.5 x i0 cells/mi. After sonication on ice the mixture was centrifuged at 500g for 10 mm (4#{176}C). The supennatant was spun at 48,000g for 10 mm (4#{176}C)and the pellet resuspended with sonication into 2 ml of 0.34 M sucrose. Protein determinations were made using the method of Lowry et al. [21].
Electron
Microscopy
Electron micrographs of 0.i-m-thick sections were obtained on a Philips EM-400 microscope operating at 80 keV. 5cctions of 1.0 im were observed using a Philips CM-30 intermediate-voltage electron microscope operating at 300 keV. The pH is 7.3 and cacodylate buffer and sucrose concentrations are 0.1 M unless stated otherwise.
Peroxidase
Staining
PMNs
fixed
were
for
10 mm
in
1%
glutaraldehyde
0504 in cacodylate buffer, dehydrated in several steps ethanol-water mixtures (25 to 100%), and then embedded Epoxy 812 (Ernest Fullam, Inc.). Control PMNs treated in the same fashion in the absence of DAB.
with in were
592
1992
of Leukocyte
Biology
Volume
Localization
pelleted glutanaldehyde giutaraldehyde cacodylate-sucrose
Tightly
cell pellets tions having
by
Using
Frozen,
Thawed
Sections
PMNs were fixed for 1.5 h with 1% in a solution of cacodylate-sucrose. Excess was removed by repeated washing in buffer. Sucrose was infiltrated into the
equilibrating increasing
the pellets concentrations
with a series of soluof sucrose in cacody-
late buffer. After freezing the pellet in liquid nitrogen, 0.1and l.0-m-thick frozen sections were cut and transferred to Fonmvan carbon-coated, 200-mesh nickel grids. The following reactions were carried out by placing the grids section side down onto drops of solution on Parafilm. All of the solutions are buffered with Gey’s buffered salt solution (GBSS) unless stated otherwise. The grids were washed with 0.1% glycine to remove excess sucrose and to block free aldehydes, treated with 2% gelatin to prevent nonspecific bonding of antibodies, rinsed with buffer, and incubated overnight at 4#{176}C with 10 g/ml rabbit antihuman lactofernin (Chappel Laboratories). After the grids were rinsed with buffer, they were incubated with a 1:1:2 solution of goat antirabbit conjugated to 15-nm gold particles, goat antimouse conjugated to 5-nm gold particles (Janssen Life Sciences Products), and GBSS. All grids were thoroughly rinsed with GBSS and cacodylate-sucrose buffer and the antibodies fixed with 1% glutaraldehyde in cacodylate-sucrose buffer. The immunoglobin-treated sections were preserved using the plastic embedding procedure of Keller et al. [i6], which was modified as follows. Excess glutaraldehyde was removed from the sections by rinsing with cacodylate-sucrose buffer and then with cacodylate buffer. The sections were postlixed with OsO4 by incubating with 0.5% 0504 in cacodylate buffer for 10 mm. Following a thorough rinse with cacodylate buffer, the grids were incubated for 20 mm in a dimple dish containing 0.5% aqueous uranyl acetate and then dehydrated with ethanol-water washes as described above. The grids were infiltrated with two changes of LR White acrylic resin (London resin), blotted between two pieces of filter paper, and then allowed to polymerize overnight at 60#{176}Cin vacuuo. The volume of PMNs occupied by beads was determined by point count steneology of sections from both Eponembedded and frozen material [33]. A grid of points spaced 0.5 cm apart was laid oven micrographs taken of sections through 50 cells and the ratio of points on cytoplasm to points on beads determined as an estimate of the volume of cytoplasm occupied by beads. In Epon-embedded material, beads were separated into those associated with myeloperoxidase activity and those that were not. In a similar manner, beads in frozen sections associated with lactoferrin were differentiated from those showing no association.
solution
buffered with cacodylate-sucrose and then washed with cacodylate-sucrose solution containing 0.1% glycine. Myeloperoxidase was localized using the diaminobenzidine (DAB) method of Graham and Karnovsky [12] as modified for use with PMNs [5]. Cells were washed for 5 mm in 0.05 M Tnis-HC1 and then incubated with DAB and H2O2 in 0.05 M Tnis-HC1 for 1 h. Excess DAB was removed by washing with 0.05 M Tris-HC1. The sample was postfixed with 1%
Journal
Lactoferrin
51, June
RESULTS When 5 x 106 PMNs are exposed to 1.2 x i0 linoleyl alcohol-coated 0.732-jm-diameten CM latex beads, 02 is consumed and H202 is released into the medium. Representative experiments are plotted as nmol (02 and H2O2) versus time in Figure 1. SOD-inhibitable fernicytochnome c reduction is not observed until between 15 and 30 mm after the addition of the beads (Fig. 2a). Preincubating PMNs for 5 mm with cytochalasin B before treating with the CM latex beads reduced the lag time (Fig. 2a). Glucose consumption rose after exposure to beads (data not shown) and there was
both with beads and with intracellular granules (Fig. 4b). The granules that stained for peroxidase had a homogeneous size with an average profile diameter of 0.36 ± 0.03 tim, consistent with their identification as azunophiic granules. Approximately 10% of the volume of the cytoplasm (exclud-
140
120
ing
I 00
the
nucleus)
of cytoplasm 80 S
: 60
40
was
occupied
occupied
by beads.
by beads
The
colocalized
volume
activity was 5.1% and therefore 51% of the bead associated with peroxidase activity. Cells stained with antilactofernin antibodies localization around the outer cell membrane and beads in activated PMNs (Fig. 4c). Both activated ing PMNs had intracellular granules that stained .lactofennin.
The
antilactofennin-staining
percent
with
penoxidase volume
is
displayed around the and restpositive for
granules
are
gener-
20
0 10 Tim.
15
20
25 S
(mm)
Fig.
1. Oxygen metabolism and metabolites from PMNs treated with x I0 0.73-timdiameter linoleyl alcohol-coated beads added at time 0. (0) Representative plot of net oxygen consumption by 9 x 106 PMNs. The background 02 consumption by resting PMNs has been subtracted. (#{149}) Representative plot of hydrogen peroxide secretion by i.5 x 106 PMNs in the presence of I mM sodium azide used to inhibit myeloperoxidase. 1
I
a time-dependent release of lysozyme into the medium (Fig. 2b). LDH levels in the medium were essentially constant (4.4 ± 1.5% of total LDH) in each of the experiments for all time points. Uncoated 0.822-tm-diameter CM latex beads gave a profile of SOD-inhibitable fernicytochnome c reduction versus time similar to that seen with cytochalasin
3 0
8
the
role
of bead
surface
in causing
O2
the concentration of beads to 2.4 x 108 particles/ml resulted in detectable levels of O2. The rate of SOD-inhibitable fennicytochnome c reduction versus the number of untreated 2.02-tm-diameten beads pen PMN is shown in Figure 3. The rates were assessed by determining the best fit of the points between 15 mm and 45 mm to a straight line. Two of the points shown in Figure 3 were taken from studies in which comparable methods were used [1, 9]. To determine whether the NADPH-dependent oxidase was activated, particulate fractions from PMNs were assayed for NADPH-dependent fennicytochrome c reduction [22]. Two different stimuli and two different experimental controls were studied. The results are reported as the mean of two determinations. PMNs (1.5 x 108) incubated at 37#{176}Cfor 15 mm either with 0.732-sm-diameter linoleyl alcohol-coated CM latex beads or with PMA gave rates of fenricytochnome c reduction of 3.7 ± 0.6 and 14.6 ± 2.3 nmol/min/mg protein, respectively. Preparations incubated for 15 mm a stimulus on incubated rates of reduction of protein, respectively.
Electron micrognaphs after treatment with CM alcohol-coated CM latex that the plasma membrane micrographs taken after penoxidases show that
at
at 0#{176}C for 15 mm with 0.6 ± 0.3 and 0.9 ± 0.5
of PMN preparations fixed 15 mm latex beads showed that the linoleyl beads had been internalized and was intact (Fig. 4a). Electron 15-mm incubations and stained for penoxidase activity was associated
Thomas
et al.
3
secre-
tion, SOD-inhibitable ferricytochrome c reduction stimulated by 2.02-jm-diameten hydrophobic latex beads was examined as a function of time (Fig. 2c). Untreated beads at 2.4 x l0 panticles/ml do not stimulate O2 secretion. However, treating the beads with human IgG or increasing
37#{176}Cwithout beads gave nmol/min/mg
0
It
B (Fig. 2a). To examine
8
C
Superoxide
S 0
0 S
0
10
20
30
Tim.
40
50
60
(mm)
Fig. 2. Time dependence of superoxide release by PMNs after treatment with different latex beads. (a) Superoxide dismutase-inhibitable reduction of ferricytochrome c versus time by 5.1 x 106 PMNs treated with I x i0 0.73-zm-diameter linoleyl alcohol-coated CM latex beads in the presence (#{149}, solid line) and in the absence (0, dot-dash line) of cytochalasin B. Data points are the averages of five and seven experiments, respectively, with at least duplicate samples at each time point for each experiment. The response of PMNs to CM latex beads (0.82 sm in diameter) that had not been coated with linoleyl alcohol (+ , dashed line) is shown for comparison. (b) Appearance of lysozyme in the medium displayed as percent of total lysozyme versus time for PMNs treated with linoleyl alcohol-coated CM latex beads (0.82 m in diameter). PMN levels were from 2 x 106 to 5.5 x I0 per cxperiment and PMNs were treated with 1 x i0 linoleyl alcohol-coated CM latex beads. The average of four experiments is shown. The dashed line without points is the time course of SOD-inhibitable ferncytochrome c reduction from (a) shown for comparison. (c) Superoxide dismutase-inhibitable reduction of ferricytochrome c versus time from 5 x 106 PMNs treated with 7.1 x 10 2.02-sm-diameter human IgG-coated latex beads (#{149}) (one experiment with duplicate samples), and 7.1 x l0 (U) or 7.1 x 108 () uncoated beads (one experiment each with duplicate samples). See Materials and Methods for details.
generation
by
human
PMN
in response
to latex
beads
593
ally smaller an average
peroxidase-staining of 0.27 ±
0.03
m,
granules and consistent
have with
their identification as specific granules. The antilactofernin study showed only 7.4% of the cytoplasm was occupied by beads. Approximately 78% ofthe beads were associated with antilactoferrin staining. Small amounts of antimouse anti-
C
E
than the diameter
IC
U
E 0 0
body were constituted preparations
0
.
found randomly distributed less than 0.1% of the total (data not shown).
on the cells, gold bound
but it to the
. 0
E a
DISCUSSION 20
40
60
80
100
120
140
In this
study
several
measures
of 02
metabolism
induced
by
been begin
corwi-
B#{149}.ds/PMN
Fig. 3. Dependence tion
on the
number
hydrophobic beads from the literature: corrected to a rate
of the
rate
of SOD-inhibitable
ferricytochrome
c reduc-
of beads
per PMN using untreated 2.02-sm-diameter 5 x 106 PMNs per tube. Two points are taken (0) from ref. i and (U) from ref. 9. These points were for 5 x 106 PMNs. See Materials and Methods for de-
(#{149}) and
tails.
linoleyl related.
alcohol-coated Both H2O2
CM secretion
4. Electron
micrographs
magnification)
CM can
15 mm
latex beads. be seen
reaction
and are
product
surrounding associated
presumably ules. tioned
(c)
after with
Biology
Volume
51, June
i992
preparations with
1 x i0
the
cell surface
(x
linoleyl
of unstained as well
as within
18,000 original alcohol-coated
PMN, the
beads
(B)
cytoplasm.
to demonstrate peroxidase localization. In order to highof myeloperoxidse, the cell was not counterstained. Dense can be seen associated with azurophiic granules (arrows) internalized beads (arrowheads). However, not all beads
with
myeloperoxidase reaction product. The unstained beads new phagosomes that have not yet ftised with granimmunologically stained to localize lactoferrin. Cryosecwere treated with rabbit antihuman lactoferrin followed by
represent PMN
PMNs
goat antirabbit antibodies be seen associated with ( arrows).
of Leukocyte
of PMN treatment
(a) In this photomicrograph
in contact
(b) PMN stained light the presence
Journal
beads have consumption
thin approximately 4 mm after exposure to the beads. Figure 1 shows that 02 uptake precedes the detection of H2O2. However, the maximum rates of the two processes correlate well with one another: 02 consumption taken from the point
Fig.
594
latex and 02
conjugated internalized
to 15-nm beads
gold particles. The as well as within
gold can granules
on the curve having the greatest slope cells/h, and in the presence of NaN3 the rate of H2O2 generation (four experiments) mmol/i01#{176} cells/h.
The
rates
of 02
is 2.1 mmol/i01#{176} average maximum is (2.7 ± 0.9)
uptake
and
H202
secre-
tion (in the presence of azide) are larger than the rates reported for 0.82-sm-diameter IgG-treated latex particles, 0.95 mmol/i01#{176} cells/h [31] and 1.2 mmol/i01#{176} cells/h [15], respectively, but comparable in magnitude to the rate of H2O2 secretion on treatment with opsonized zymosan (from Fig. 2 of ref. 28), 2.1 mmol/i01#{176} cells/h. Direct comparisons with other reported results are difficult because the rates are influenced by the bead-to-PMN ratio. Degranulation, measured by the appearance in the medium of lysozyme, an enzyme found in both the specific and azurophilic granules, begins immediately and increases rapidly for 30 mm. Electron microscopy combined with cytochemical analysis confirmed that both the azurophilic and specific granules released their contents into the phagosome within the first 15 mm after exposure to CM latex beads. These studies are consistent with earlier reports showing that peroxidase activity was localized around the bead vacuoles [3, 7] and that lactofernin was located in the vacuole and on the outer PMN membrane [19]. About 90% of the degnanulated lactofennin is reported to be found in the medium with the balance associated with the phagosome [19]. We did not quantify the distribution of lactofernin, but we have demonstrated that a significant amount is found in the phagosome, in support of the earlier conclusions. Neutrophil NADPH-dependent oxidase is active 15 mm after treating the PMNs with linoleyl alcohol-coated CM latex beads, but O2 cannot be detected in the medium. NADPH-dependent oxidase activity was approximately equal to that reported for 5 M A23i87 in the absence of cytochalasin B [22] but lower than the activity obtained with PMA. The magnitude of the response is similar to that found in the
for C5a presence
and N-fonmyl-methionyl-leucyl-phenylalanine of cytochalasin B [22]. The delayed
onset
of
O2 release can be reversed by pretreatment of the PMNs with cytochalasin B. Since cytochalasin B inhibits phagolysosome formation [26], the early absence of extracellulan 02 with linoleyl alcohol-coated CM latex beads may be due to the formation of a tightly sealed phagolysosome. Identification of an activated NADPH-dependent oxidase after treatment with linoleyl alcohol-coated CM latex beads is conclusive evidence that O2 was produced by the PMNs at time points during which O2 cannot be detected in the medium. The formation of a tightly sealed phagolysosome excludes fernicytochnome c from the phagolysosome, while the phagolysosome membrane acts as an effective barrier to the diffusion of O2 through the cytoplasm into the medium. The conjugate acid of 02, HO2 , could diffuse through the membrane, but it would be readily scavenged by cytoplasmic SOD [iO]. If the formation of a tightly sealed phagolysosome prevents leakage of O2 into the medium, what is the cause of the detection of O2 after 30 mm when the beads are coated with linoleyl alcohol? It is unlikely that leakage of O2 from the phagolysosomes can explain the detection of O2 at the later time points for two reasons: (1) the LDH level in the medium at 60 mm is not significantly different from that found at either 5 or 15 mm, and (2) as illustrated in Figure 2b, lysozyme release does not show the same profile as the appearance of O2 in the medium. These observations suggest that active NADPH-dependent oxidase appears on the external plasma membrane as a result of either activation of fresh oxidase or fusion of phagolysosome membrane with the
Thomac
et al.
Superoxide
outer membrane followed by movement to the outer membrane surface. The report of Green et al. [14] provides
of activated an
oxidase
explanation
for
the behavior of PMNs treated with latex beads. In their study with IgG-treated latex beads, 02 consumption was shown to be linearly related to the bead-to-PMN ratio but the secretion of O2 could not detected until a threshold bead-to-PMN ratio was attained. They suggested that when PMNs are exposed to large numbers of particles they attempt to phagocytose additional first-formed phagocytic vesicles thereby allowing O2 to escape hypothesis is supported by a study in which the amount of O( detected versely correlated to the ability of
beads rapidly before the have completely closed, into the medium. Their of liposome phagocytosis in the medium was inthe PMNs to internalize
the liposomes [23]. When PMNs were treated with 2.02-smdiameter hydrophobic latex beads, 02 was detected in the medium at the larger bead-to-PMN ratios, as shown in Figures 2c and 3. Two of the points shown in Figure 3 were taken from studies [i, 9] that used beads of different diameters. Considering the potential errors, the correlation is quite good. Therefore, it can be concluded that the secretion of O2 by PMNs treated with hydrophobic latex beads is qualitatively similar to that by PMNs treated with IgGcoated latex beads [14], but with the hydrophobic beads a larger bead-to-PMN ratio is required before 02 is detected in the medium. Consistent with the assumption of a qualitative similarity in the response to IgG-coated and uncoated latex beads, 1.7 x i0 IgG-coated hydrophobic beads cause greater O2 secretion than a similar number of uncoated beads (Fig. 2c). However, coating CM latex beads with linoleyl alcohol seems to have the opposite effect, delaying the detection of O2 when compared to uncoated CM latex beads (Fig. 2a). Therefore, within any group of particles both the magnitude of the stimulation induced by the particle and the ease of phagolysosome formation are factors that determine the minimum number ofparticles that give detectable levels of O2. The dependence of O( secretion on the bead-to-PMN ratio implies that the detection of 02 in the medium is a function of the physical constraints on phagolysosome formation. However, an alternative suggestion for the lack of detectable O2 is that there is a second mechanism for H2O2 formation in which 02 undergoes a direct two-electron reduction to H202. The demonstration of an active NADPH-dependent oxidase when no O2 can be detected does not support the postulate of a second O2-reducing system as an explanation for our observations. In conclusion, CM latex beads with and without a coat of linoleyl alcohol appear to cause 02 reduction by the PMNs, as has been reported for IgGand serum-treated latex partides [1, 2, 14, 30, 32]. Coating CM latex beads with linoleyl alcohol causes a pronounced lag between the initial treatment with beads and the detection ofO2. However, since the NADPH-dependent oxidase is active during the period when 02 cannot be detected, linoleyl alcohol does not appear to change the mechanism of 02 reduction. Frustrating phagocytosis by pretreatment with cytochalasin B resulted in immediate release of O2. These results suggest that it is the formation of a tightly sealed phagolysosome that is responsible for the absence of detectable 02 when PMNs are treated with linoleyl alcohol-coated CM latex beads. The appearance of extracellular O2 after activation of NADPH-dependent tennal membrane.
generation
by human
PMN
30 mm may oxidase
in response
be due located
to latex
beads
to the late on the cx-
595
improved
ACKNOWLEDGMENTS
procedure
plastic
We thank Drs. L.C. McPhail and J.T. O’Flaherty for cnitically reviewing the manuscript and Ms. Dorothy Harris for her technical assistance. This work was supported by National Institutes of Health grant GM 29611. The electron microscopy was supported in part by National Resource Intermediate Voltage Electron Microscope grant RR-02722.
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J.W.
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by
human and
Randall,
ofthe
J.
R.J.
j
30.
31.
EL. Inhibition to chemotactic
of in factors
respiratory
by 72,
phagocyto-
neutrophils
vivo and in by a competitive
Biol.
leukocytes Clin. Invest.
Antibody-dependent human
polymor-
reagent.
Activation
polymorphonuclear soluble stimuli.
McCall,
adenosine
is dependent
on the physical state of the liposomal membrane. Biochim. phys. Acta. 692, 101, 1982. 24. O’Flaherty, J.T., Showell, H.J., Kreutzer, DL., Ward, PA.,
in 254,
oxidative 3, i,
ultrathin
USA
DeChatelet,
sis of haptenated
25.
Karnovsky,
McPhail,
Sd.
of quercetin
burst enzyme chemoattractants 192, 1983. 23. Munn, MW.,
1975.
8. Cheson, killing
Lowry,
Protein Chem.
and
hydrogen peroxide j Cell Bid. 64,
GD.,
Effects
phonuclear
study
demonstration of leukocyte phagosomes.
Long,
Acad.
triphosphatase
of the production of superoxide radicals by neutrophil NADPH oxidase. FEBS Lett. 145, 323, 1982. 5. Bass, D.A., Lewis, J.C., Szejda, P., Cowley, L., and McCall, C.E. Activation of lysosomal acid phosphatase of eosinophil leukocytes. Lab. Invest. 44, 403, 1981. 6. Bergmeyer, H.U., and Bernt, E. Lactate dehydrogenase. In Methods in Enzymatic Analysis (Bergmeyer, H.U., Ed.) New York: Academic Press, p. 574, 1974. 7. Briggs,
NatI.
CE.
Kipnes,
J.V.,
Rossi,
microscopy:
of immunolabeled
81, 5744, 1984. 17. Klebanoff, S.J., and Clark, R.A. The Neutrophil: Function and Clinical Disorders. New York: North-Holland, pp. 294-295, 1978. 18. Kvarstein, B. Oxygen consumption during the initial stage of human leucocyte phagocytosis of polystyrene latex particles. Scand. J. Clin. Lab. Invest. 25, 337, i970. 19. Leffell, MS., and Spitznagel, J.K. Fate of human lactofernin and myeloperoxidase in phagocytizing human neutrophils: effects of immunoglobulin G subclasses and immune complexes coated on latex beads. Infect. Immun. i2, 813, 1975. Proc.
20.
R.S., and Curnutte, JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. j Clin. Invest. 52, 741, 1973. 2. Baehner, R.L. , Gilman, N., and Karnovsky, ML. Respiration and glucose oxidation in human and guinea pig leukocytes: comparative studies. j Clin. Invest. 49, 692, 1970. 3. Baehner, R.L., Karnovsky, M.J., and Karnovsky, ML. Degranulation of leukocytes in chronic granulomatous disease. j Clin. Invest. 48, 187, 1969. 1. Babior,
for immunoelectron
embedding
vitro
neutrophil antagonist.
Bioand j
Immunol. 120, 1326, 1978. O’Flaherty, J.T., Wykle, R.L., Lees, C.J., Shewmake, T., McCall, C.E., and Thomas, M.J. Neutrophil-degranulating action of 5,i2-dihydroxy-6,8,iO,i4-eicosatetraenoic acid and 1-0alkyl-2-acetyl-sn-glycero-3-phosphocholine. Am. j Pathol. 105, 264, 1981. Oliver, J.M. Cell biology of leukocyte abnormalitiesmembrane and cytoskeletal function in normal and defective cells. Am. j Pathol. 93, 221, 1978. Root, R.K., Metcalf, J., Oshino, N., and Chance, B. H202 release from human granulocytes during phagocytosis. j Clin. Invest. 55, 945, 1975. Test, ST., and Weiss, S.J. Quantitative and temporal charactenization of the extracellular H202 pool generated by human neutrophils. j BioL Chem. 259, 399, 1984. Thomas, M.J., Shirley, P.S., Hednick, C., and DeChatelet, L.R. Role of free radical processes in stimulated human polymorphonuclear leukocytes. Biochemistry 25, 8042, 1986. Tsan, M.-F., and McIntyre, PA. The requirement for membrane sialic acid in the stimulation of superoxide production during phagocytosis by human polymorphonuclear leukocytes. j Exp. Med. 143, 1308, 1976. Weening, R.S., Roos, D., and Loos, J.A. Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. j Lab. Clin. Med. 83, 570, 1974.
32. Weening, R.S., the production
Wever, R., and Roos, D. Quantitative aspects of of superoxide radicals by phagocytizing human granulocytes. j Lab. Clin. Med. 85, 245, 1975. 33. Weibel, ER., Kistler, G.S., and Scherle, W.F. Practical stereological methods for morphometric cytology. j Cell Biol. 30, 23, 1966.
of Biochemisty*
by the human to latex beads
Catherine and Pamela
C. Hedrick7
and
The
Pathology,
Wake
posure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c. J. Leukoc. Biol. Si: 591-596; 1992. Words:
hydrogen
peroxide
.
phagocytosis
respiratory
burst
INTRODUCTION The formation of O2 by polymorphonuclear leukocytes (PMNs) was first reported by Babion et al. [1] after treating PMNs with latex beads and assessing O2 secretion by the reduction of fernicytochnome c. Studies have repeatedly confirmed [17] the original observation, and O2 has been identified using spin-trapping techniques [4, 13]. Prior to the detection of O2, only H202 formation [3] had been linked to the increased 02 consumption [18, 31] by activated PMNs. Several studies have shown that a variety of actions are stimulated when PMNs are treated with 1.2 to 0.8 jm diameten hydrophobic beads that have been coated with serum or with immunoglobulin G (IgG). Large increases in the rates of glucose oxidation, 02 consumption, H2O2 fonmation, and O2 release have been reported [1, 3, 8, 9, ii, 14, 15, 18, 27, 31, 32]. Untreated hydrophobic latex beads are reported to elicit most ofthe responses obtained with opsonization [2, 9]. However, the question of whether hydrophobic beads cause O2 secretion is disputed. Some laboratories report superoxide dismutase (SOD)-inhibitable fernicytochrome c reduction [1, 2, 30], whereas others were unable to detect significant amounts of O2 [9]. We have used latex beads as a vehicle to study the oxidant chemistry of the phagolysosome [29]. The latex beads carry probe molecules, generally linoleyl alcohol, into the phagolysosome to detect and quantitate oxidants like HO 102, and HOC1. Because O2 generation is not reported after treatment with certain types of latex beads, it was necessary to establish the response modified (CM) latex bead course of these investigations
of the PMN used in these we examined
to
Sharon
Smith
Jian
Pang7
W. Gray
Jerome,t
S. Shirley*
Abstract: In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxymodified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 mm after the initial exposure to beads. However, an 02-producing, NADPH-dependent oxidase is active iS mm after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after cx-
Key
polymorphonuclear
the carboxystudies. In the the response to
Forest
University
Medical
Center,
Winston-Salem,
North
Carolina
CM latex beads coated with linoleyl alcohol by measuring 02 and H2O2 secretion, 02 consumption, NADPHdependent oxidase activity, and the release of lactofennin and myeloperoxidase from granules. We found a significant lag between the initial treatment with beads and the detection of O2, but the other measures process. Our studies imply same physiologic responses cytic
indicated a normal phagocytic that latex particles induce the that characterize other phago-
particles.
MATERIALS
AND
METHODS
General Polystyrene canboxylate-modified hydrophobic latexes (0.732 and 0.822 ,zm in diameter) were obtained from Interfacial Dynamics Corporation. Polyvinyltoluene latex beads (Dow, 2.02 m in diameter) were obtained from Duke Scientific Co. Dulbeccds phosphate-buffered saline (PBS) was from Grand Island Biological Co. Dextran T 500 was from Pharmacia. Fernicytochrome c, phonbol mynistate acetate (PMA), cytochalasin B, NADH, sodium pyruvate, micrococcus lysodeikticus,’ human IgG, and SOD were from Sigma. Linoleyl alcohol was from NuChek Prep., Inc.
Assay
Procedures
PMNs were isolated as previously described [29]. This method yields a preparation that contains greater than 87% granulocytes (generally greater than 95%) with a viability greater than 95% as judged by trypan blue exclusion. Linoleyl alcohol-coated latex beads were prepared as described [29]. IgG-coated beads were prepared by allowing the latex beads to stand overnight at 4#{176}C with 30 mg/mI IgG and then washing the beads five times with deionized water. Oxygen uptake was monitored with a YSI model 53 oximeter using a Clark electrode. A YSI model 25 oxidase meter equipped with a YSI model 2510 oxidase probe was used to measure H2O2 secretion [28]. Hexose monophosphate shunt activity was assessed using the procedure of Long et al. [20]. Degranulation was assessed by the percentage of total lysozyme released into the medium [24, 25]. Cell viability was determined by measuring the percentage of total lactate dehydrogenase (LDH) released by the PMNs [6, 25]. Duplicate
Abbreviations: Gey’s buffered hydrogenase; tate; PMN,
CM, carboxy-modified; DAB, diaminobenzidine; GBSS, salt solution; IgG, immunoglobulin G; LDH, lactate dePBS, phosphate-buffered saline; PMA, phorbol myristate acepolymorphonudear leukocyte; SOD, superoxide dismutase.
Reprint requests: Michael J. Thomas, Department Wake Forest University Medical Center, Medical Winston-Salem, NC 27157-1016. Received September 17, 1991; accepted December
Journal
of Leukocyte
Biology
Volume
of Biochemistry, The Center Boulevard, 13,
51, June
1991.
1992
591
determinations lysozyme and Superoxide SOD-inhibitable
were obtained LDH. production was reduction
at
each
time
point
for
determined by measuring of fernicytochrome
both the [1].
c
Specifically, tubes were prepared on ice containing 1.7 x i06 PMN/ml, 433 ig/ml fennicytochnome c, and 1.1 mM glucose in PBS with a total volume of 3 ml. Half of the tubes also contained 10 g/ml SOD. Control tubes were kept on ice during the experiment. The remaining tubes were incubated for 5 mm at 37#{176}Cand then treated with 100 tl of bead solution. Samples (duplicate or triplicate) were removed at specific intervals, chilled on ice, and then either filtered through 0.45-tim filters or centrifuged at 15,000g before measuring the optical absonptions at 550 and 556 nm. When cytochalasin for 5 mm 1.7
x
B was with 12.5 PMN/ml
106
used, tg/ml (4.2
5 x 106 PMNs were preincubated cytochalasin B and then diluted g/ml cytochalasin B) for the
to cx-
peniment. The activity of the NADP-dependent oxidase was measured in particulate fractions by the method of McPhail and Snyderman [22] without cytochalasin B. Solutions were prepared on ice containing 1.5 x 108 PMNs in 3 ml PBS and incubated for a total of 20 mm. The following treatments were evaluated: (1) incubation on ice with the addition of 50 l linoleyl alcohol-coated CM latex beads (5 x i0 beads/ml) 5 mm after the start of the experiment; (2) incubation at 37#{176}Cfor 5 mm followed by treatment with 50 l of beads; (3) incubation at 37#{176}Cwithout beads; and (4) incubation for 5 mm at 37#{176}Cfollowed by treatment with 1 l PMA (33 ng/ml final concentration). All samples were treated with 30 jl of sodium azide 4 mm after the start of the experiment. At the end of the incubation period the tubes were treated with 15 ml ice-cold PBS and placed on ice. PMNs were pelleted at l8Og (4#{176}C).The pellet was resuspended in 0.34 M sucrose to a concentration of 2.5 x i0 cells/mi. After sonication on ice the mixture was centrifuged at 500g for 10 mm (4#{176}C). The supennatant was spun at 48,000g for 10 mm (4#{176}C)and the pellet resuspended with sonication into 2 ml of 0.34 M sucrose. Protein determinations were made using the method of Lowry et al. [21].
Electron
Microscopy
Electron micrographs of 0.i-m-thick sections were obtained on a Philips EM-400 microscope operating at 80 keV. 5cctions of 1.0 im were observed using a Philips CM-30 intermediate-voltage electron microscope operating at 300 keV. The pH is 7.3 and cacodylate buffer and sucrose concentrations are 0.1 M unless stated otherwise.
Peroxidase
Staining
PMNs
fixed
were
for
10 mm
in
1%
glutaraldehyde
0504 in cacodylate buffer, dehydrated in several steps ethanol-water mixtures (25 to 100%), and then embedded Epoxy 812 (Ernest Fullam, Inc.). Control PMNs treated in the same fashion in the absence of DAB.
with in were
592
1992
of Leukocyte
Biology
Volume
Localization
pelleted glutanaldehyde giutaraldehyde cacodylate-sucrose
Tightly
cell pellets tions having
by
Using
Frozen,
Thawed
Sections
PMNs were fixed for 1.5 h with 1% in a solution of cacodylate-sucrose. Excess was removed by repeated washing in buffer. Sucrose was infiltrated into the
equilibrating increasing
the pellets concentrations
with a series of soluof sucrose in cacody-
late buffer. After freezing the pellet in liquid nitrogen, 0.1and l.0-m-thick frozen sections were cut and transferred to Fonmvan carbon-coated, 200-mesh nickel grids. The following reactions were carried out by placing the grids section side down onto drops of solution on Parafilm. All of the solutions are buffered with Gey’s buffered salt solution (GBSS) unless stated otherwise. The grids were washed with 0.1% glycine to remove excess sucrose and to block free aldehydes, treated with 2% gelatin to prevent nonspecific bonding of antibodies, rinsed with buffer, and incubated overnight at 4#{176}C with 10 g/ml rabbit antihuman lactofernin (Chappel Laboratories). After the grids were rinsed with buffer, they were incubated with a 1:1:2 solution of goat antirabbit conjugated to 15-nm gold particles, goat antimouse conjugated to 5-nm gold particles (Janssen Life Sciences Products), and GBSS. All grids were thoroughly rinsed with GBSS and cacodylate-sucrose buffer and the antibodies fixed with 1% glutaraldehyde in cacodylate-sucrose buffer. The immunoglobin-treated sections were preserved using the plastic embedding procedure of Keller et al. [i6], which was modified as follows. Excess glutaraldehyde was removed from the sections by rinsing with cacodylate-sucrose buffer and then with cacodylate buffer. The sections were postlixed with OsO4 by incubating with 0.5% 0504 in cacodylate buffer for 10 mm. Following a thorough rinse with cacodylate buffer, the grids were incubated for 20 mm in a dimple dish containing 0.5% aqueous uranyl acetate and then dehydrated with ethanol-water washes as described above. The grids were infiltrated with two changes of LR White acrylic resin (London resin), blotted between two pieces of filter paper, and then allowed to polymerize overnight at 60#{176}Cin vacuuo. The volume of PMNs occupied by beads was determined by point count steneology of sections from both Eponembedded and frozen material [33]. A grid of points spaced 0.5 cm apart was laid oven micrographs taken of sections through 50 cells and the ratio of points on cytoplasm to points on beads determined as an estimate of the volume of cytoplasm occupied by beads. In Epon-embedded material, beads were separated into those associated with myeloperoxidase activity and those that were not. In a similar manner, beads in frozen sections associated with lactoferrin were differentiated from those showing no association.
solution
buffered with cacodylate-sucrose and then washed with cacodylate-sucrose solution containing 0.1% glycine. Myeloperoxidase was localized using the diaminobenzidine (DAB) method of Graham and Karnovsky [12] as modified for use with PMNs [5]. Cells were washed for 5 mm in 0.05 M Tnis-HC1 and then incubated with DAB and H2O2 in 0.05 M Tnis-HC1 for 1 h. Excess DAB was removed by washing with 0.05 M Tris-HC1. The sample was postfixed with 1%
Journal
Lactoferrin
51, June
RESULTS When 5 x 106 PMNs are exposed to 1.2 x i0 linoleyl alcohol-coated 0.732-jm-diameten CM latex beads, 02 is consumed and H202 is released into the medium. Representative experiments are plotted as nmol (02 and H2O2) versus time in Figure 1. SOD-inhibitable fernicytochnome c reduction is not observed until between 15 and 30 mm after the addition of the beads (Fig. 2a). Preincubating PMNs for 5 mm with cytochalasin B before treating with the CM latex beads reduced the lag time (Fig. 2a). Glucose consumption rose after exposure to beads (data not shown) and there was
both with beads and with intracellular granules (Fig. 4b). The granules that stained for peroxidase had a homogeneous size with an average profile diameter of 0.36 ± 0.03 tim, consistent with their identification as azunophiic granules. Approximately 10% of the volume of the cytoplasm (exclud-
140
120
ing
I 00
the
nucleus)
of cytoplasm 80 S
: 60
40
was
occupied
occupied
by beads.
by beads
The
colocalized
volume
activity was 5.1% and therefore 51% of the bead associated with peroxidase activity. Cells stained with antilactofernin antibodies localization around the outer cell membrane and beads in activated PMNs (Fig. 4c). Both activated ing PMNs had intracellular granules that stained .lactofennin.
The
antilactofennin-staining
percent
with
penoxidase volume
is
displayed around the and restpositive for
granules
are
gener-
20
0 10 Tim.
15
20
25 S
(mm)
Fig.
1. Oxygen metabolism and metabolites from PMNs treated with x I0 0.73-timdiameter linoleyl alcohol-coated beads added at time 0. (0) Representative plot of net oxygen consumption by 9 x 106 PMNs. The background 02 consumption by resting PMNs has been subtracted. (#{149}) Representative plot of hydrogen peroxide secretion by i.5 x 106 PMNs in the presence of I mM sodium azide used to inhibit myeloperoxidase. 1
I
a time-dependent release of lysozyme into the medium (Fig. 2b). LDH levels in the medium were essentially constant (4.4 ± 1.5% of total LDH) in each of the experiments for all time points. Uncoated 0.822-tm-diameter CM latex beads gave a profile of SOD-inhibitable fernicytochnome c reduction versus time similar to that seen with cytochalasin
3 0
8
the
role
of bead
surface
in causing
O2
the concentration of beads to 2.4 x 108 particles/ml resulted in detectable levels of O2. The rate of SOD-inhibitable fennicytochnome c reduction versus the number of untreated 2.02-tm-diameten beads pen PMN is shown in Figure 3. The rates were assessed by determining the best fit of the points between 15 mm and 45 mm to a straight line. Two of the points shown in Figure 3 were taken from studies in which comparable methods were used [1, 9]. To determine whether the NADPH-dependent oxidase was activated, particulate fractions from PMNs were assayed for NADPH-dependent fennicytochrome c reduction [22]. Two different stimuli and two different experimental controls were studied. The results are reported as the mean of two determinations. PMNs (1.5 x 108) incubated at 37#{176}Cfor 15 mm either with 0.732-sm-diameter linoleyl alcohol-coated CM latex beads or with PMA gave rates of fenricytochnome c reduction of 3.7 ± 0.6 and 14.6 ± 2.3 nmol/min/mg protein, respectively. Preparations incubated for 15 mm a stimulus on incubated rates of reduction of protein, respectively.
Electron micrognaphs after treatment with CM alcohol-coated CM latex that the plasma membrane micrographs taken after penoxidases show that
at
at 0#{176}C for 15 mm with 0.6 ± 0.3 and 0.9 ± 0.5
of PMN preparations fixed 15 mm latex beads showed that the linoleyl beads had been internalized and was intact (Fig. 4a). Electron 15-mm incubations and stained for penoxidase activity was associated
Thomas
et al.
3
secre-
tion, SOD-inhibitable ferricytochrome c reduction stimulated by 2.02-jm-diameten hydrophobic latex beads was examined as a function of time (Fig. 2c). Untreated beads at 2.4 x l0 panticles/ml do not stimulate O2 secretion. However, treating the beads with human IgG or increasing
37#{176}Cwithout beads gave nmol/min/mg
0
It
B (Fig. 2a). To examine
8
C
Superoxide
S 0
0 S
0
10
20
30
Tim.
40
50
60
(mm)
Fig. 2. Time dependence of superoxide release by PMNs after treatment with different latex beads. (a) Superoxide dismutase-inhibitable reduction of ferricytochrome c versus time by 5.1 x 106 PMNs treated with I x i0 0.73-zm-diameter linoleyl alcohol-coated CM latex beads in the presence (#{149}, solid line) and in the absence (0, dot-dash line) of cytochalasin B. Data points are the averages of five and seven experiments, respectively, with at least duplicate samples at each time point for each experiment. The response of PMNs to CM latex beads (0.82 sm in diameter) that had not been coated with linoleyl alcohol (+ , dashed line) is shown for comparison. (b) Appearance of lysozyme in the medium displayed as percent of total lysozyme versus time for PMNs treated with linoleyl alcohol-coated CM latex beads (0.82 m in diameter). PMN levels were from 2 x 106 to 5.5 x I0 per cxperiment and PMNs were treated with 1 x i0 linoleyl alcohol-coated CM latex beads. The average of four experiments is shown. The dashed line without points is the time course of SOD-inhibitable ferncytochrome c reduction from (a) shown for comparison. (c) Superoxide dismutase-inhibitable reduction of ferricytochrome c versus time from 5 x 106 PMNs treated with 7.1 x 10 2.02-sm-diameter human IgG-coated latex beads (#{149}) (one experiment with duplicate samples), and 7.1 x l0 (U) or 7.1 x 108 () uncoated beads (one experiment each with duplicate samples). See Materials and Methods for details.
generation
by
human
PMN
in response
to latex
beads
593
ally smaller an average
peroxidase-staining of 0.27 ±
0.03
m,
granules and consistent
have with
their identification as specific granules. The antilactofernin study showed only 7.4% of the cytoplasm was occupied by beads. Approximately 78% ofthe beads were associated with antilactoferrin staining. Small amounts of antimouse anti-
C
E
than the diameter
IC
U
E 0 0
body were constituted preparations
0
.
found randomly distributed less than 0.1% of the total (data not shown).
on the cells, gold bound
but it to the
. 0
E a
DISCUSSION 20
40
60
80
100
120
140
In this
study
several
measures
of 02
metabolism
induced
by
been begin
corwi-
B#{149}.ds/PMN
Fig. 3. Dependence tion
on the
number
hydrophobic beads from the literature: corrected to a rate
of the
rate
of SOD-inhibitable
ferricytochrome
c reduc-
of beads
per PMN using untreated 2.02-sm-diameter 5 x 106 PMNs per tube. Two points are taken (0) from ref. i and (U) from ref. 9. These points were for 5 x 106 PMNs. See Materials and Methods for de-
(#{149}) and
tails.
linoleyl related.
alcohol-coated Both H2O2
CM secretion
4. Electron
micrographs
magnification)
CM can
15 mm
latex beads. be seen
reaction
and are
product
surrounding associated
presumably ules. tioned
(c)
after with
Biology
Volume
51, June
i992
preparations with
1 x i0
the
cell surface
(x
linoleyl
of unstained as well
as within
18,000 original alcohol-coated
PMN, the
beads
(B)
cytoplasm.
to demonstrate peroxidase localization. In order to highof myeloperoxidse, the cell was not counterstained. Dense can be seen associated with azurophiic granules (arrows) internalized beads (arrowheads). However, not all beads
with
myeloperoxidase reaction product. The unstained beads new phagosomes that have not yet ftised with granimmunologically stained to localize lactoferrin. Cryosecwere treated with rabbit antihuman lactoferrin followed by
represent PMN
PMNs
goat antirabbit antibodies be seen associated with ( arrows).
of Leukocyte
of PMN treatment
(a) In this photomicrograph
in contact
(b) PMN stained light the presence
Journal
beads have consumption
thin approximately 4 mm after exposure to the beads. Figure 1 shows that 02 uptake precedes the detection of H2O2. However, the maximum rates of the two processes correlate well with one another: 02 consumption taken from the point
Fig.
594
latex and 02
conjugated internalized
to 15-nm beads
gold particles. The as well as within
gold can granules
on the curve having the greatest slope cells/h, and in the presence of NaN3 the rate of H2O2 generation (four experiments) mmol/i01#{176} cells/h.
The
rates
of 02
is 2.1 mmol/i01#{176} average maximum is (2.7 ± 0.9)
uptake
and
H202
secre-
tion (in the presence of azide) are larger than the rates reported for 0.82-sm-diameter IgG-treated latex particles, 0.95 mmol/i01#{176} cells/h [31] and 1.2 mmol/i01#{176} cells/h [15], respectively, but comparable in magnitude to the rate of H2O2 secretion on treatment with opsonized zymosan (from Fig. 2 of ref. 28), 2.1 mmol/i01#{176} cells/h. Direct comparisons with other reported results are difficult because the rates are influenced by the bead-to-PMN ratio. Degranulation, measured by the appearance in the medium of lysozyme, an enzyme found in both the specific and azurophilic granules, begins immediately and increases rapidly for 30 mm. Electron microscopy combined with cytochemical analysis confirmed that both the azurophilic and specific granules released their contents into the phagosome within the first 15 mm after exposure to CM latex beads. These studies are consistent with earlier reports showing that peroxidase activity was localized around the bead vacuoles [3, 7] and that lactofernin was located in the vacuole and on the outer PMN membrane [19]. About 90% of the degnanulated lactofennin is reported to be found in the medium with the balance associated with the phagosome [19]. We did not quantify the distribution of lactofernin, but we have demonstrated that a significant amount is found in the phagosome, in support of the earlier conclusions. Neutrophil NADPH-dependent oxidase is active 15 mm after treating the PMNs with linoleyl alcohol-coated CM latex beads, but O2 cannot be detected in the medium. NADPH-dependent oxidase activity was approximately equal to that reported for 5 M A23i87 in the absence of cytochalasin B [22] but lower than the activity obtained with PMA. The magnitude of the response is similar to that found in the
for C5a presence
and N-fonmyl-methionyl-leucyl-phenylalanine of cytochalasin B [22]. The delayed
onset
of
O2 release can be reversed by pretreatment of the PMNs with cytochalasin B. Since cytochalasin B inhibits phagolysosome formation [26], the early absence of extracellulan 02 with linoleyl alcohol-coated CM latex beads may be due to the formation of a tightly sealed phagolysosome. Identification of an activated NADPH-dependent oxidase after treatment with linoleyl alcohol-coated CM latex beads is conclusive evidence that O2 was produced by the PMNs at time points during which O2 cannot be detected in the medium. The formation of a tightly sealed phagolysosome excludes fernicytochnome c from the phagolysosome, while the phagolysosome membrane acts as an effective barrier to the diffusion of O2 through the cytoplasm into the medium. The conjugate acid of 02, HO2 , could diffuse through the membrane, but it would be readily scavenged by cytoplasmic SOD [iO]. If the formation of a tightly sealed phagolysosome prevents leakage of O2 into the medium, what is the cause of the detection of O2 after 30 mm when the beads are coated with linoleyl alcohol? It is unlikely that leakage of O2 from the phagolysosomes can explain the detection of O2 at the later time points for two reasons: (1) the LDH level in the medium at 60 mm is not significantly different from that found at either 5 or 15 mm, and (2) as illustrated in Figure 2b, lysozyme release does not show the same profile as the appearance of O2 in the medium. These observations suggest that active NADPH-dependent oxidase appears on the external plasma membrane as a result of either activation of fresh oxidase or fusion of phagolysosome membrane with the
Thomac
et al.
Superoxide
outer membrane followed by movement to the outer membrane surface. The report of Green et al. [14] provides
of activated an
oxidase
explanation
for
the behavior of PMNs treated with latex beads. In their study with IgG-treated latex beads, 02 consumption was shown to be linearly related to the bead-to-PMN ratio but the secretion of O2 could not detected until a threshold bead-to-PMN ratio was attained. They suggested that when PMNs are exposed to large numbers of particles they attempt to phagocytose additional first-formed phagocytic vesicles thereby allowing O2 to escape hypothesis is supported by a study in which the amount of O( detected versely correlated to the ability of
beads rapidly before the have completely closed, into the medium. Their of liposome phagocytosis in the medium was inthe PMNs to internalize
the liposomes [23]. When PMNs were treated with 2.02-smdiameter hydrophobic latex beads, 02 was detected in the medium at the larger bead-to-PMN ratios, as shown in Figures 2c and 3. Two of the points shown in Figure 3 were taken from studies [i, 9] that used beads of different diameters. Considering the potential errors, the correlation is quite good. Therefore, it can be concluded that the secretion of O2 by PMNs treated with hydrophobic latex beads is qualitatively similar to that by PMNs treated with IgGcoated latex beads [14], but with the hydrophobic beads a larger bead-to-PMN ratio is required before 02 is detected in the medium. Consistent with the assumption of a qualitative similarity in the response to IgG-coated and uncoated latex beads, 1.7 x i0 IgG-coated hydrophobic beads cause greater O2 secretion than a similar number of uncoated beads (Fig. 2c). However, coating CM latex beads with linoleyl alcohol seems to have the opposite effect, delaying the detection of O2 when compared to uncoated CM latex beads (Fig. 2a). Therefore, within any group of particles both the magnitude of the stimulation induced by the particle and the ease of phagolysosome formation are factors that determine the minimum number ofparticles that give detectable levels of O2. The dependence of O( secretion on the bead-to-PMN ratio implies that the detection of 02 in the medium is a function of the physical constraints on phagolysosome formation. However, an alternative suggestion for the lack of detectable O2 is that there is a second mechanism for H2O2 formation in which 02 undergoes a direct two-electron reduction to H202. The demonstration of an active NADPH-dependent oxidase when no O2 can be detected does not support the postulate of a second O2-reducing system as an explanation for our observations. In conclusion, CM latex beads with and without a coat of linoleyl alcohol appear to cause 02 reduction by the PMNs, as has been reported for IgGand serum-treated latex partides [1, 2, 14, 30, 32]. Coating CM latex beads with linoleyl alcohol causes a pronounced lag between the initial treatment with beads and the detection ofO2. However, since the NADPH-dependent oxidase is active during the period when 02 cannot be detected, linoleyl alcohol does not appear to change the mechanism of 02 reduction. Frustrating phagocytosis by pretreatment with cytochalasin B resulted in immediate release of O2. These results suggest that it is the formation of a tightly sealed phagolysosome that is responsible for the absence of detectable 02 when PMNs are treated with linoleyl alcohol-coated CM latex beads. The appearance of extracellular O2 after activation of NADPH-dependent tennal membrane.
generation
by human
PMN
30 mm may oxidase
in response
be due located
to latex
beads
to the late on the cx-
595
improved
ACKNOWLEDGMENTS
procedure
plastic
We thank Drs. L.C. McPhail and J.T. O’Flaherty for cnitically reviewing the manuscript and Ms. Dorothy Harris for her technical assistance. This work was supported by National Institutes of Health grant GM 29611. The electron microscopy was supported in part by National Resource Intermediate Voltage Electron Microscope grant RR-02722.
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