Conkling. P.R., and Sagone. ir, AL. Activation of monocyte and granulocyte antibody-dependent cytotoxicity by phorbol myristate acetate. Infect. Immun. 35.818.
Journal
Receptor Binding Neutrophils MR.
Shalaby,
MA.
Departments Molecular
of Leukocyte
Biology
41:196-204
(1987)
and Activation of Polymorphonuclear by Tumor Necrosis Factor-Alpha Palladino, Jr, S.E. Hlrabayashi, T.E. Eessalu, H.M. Shepard, and B.B. Aggarwal
of Pharmacological Biology
(TEE..
Sciences B.B.A.).
MAP..
(MRS..
Genenlech.
Inc.,
The interaction of highly purified recombinant a) with human polymorphonuclear neutrophils rTNF.a to PMN reached maximum levels in 30 analysis of competitive binding data indicated
South
S.E.H.. San
G.D.
H.M.S.),
G.D.L..
Francisco.
Lewis,
and
California
human tumor necrosis factor-alpha (rTNF. (PMN5) was investigated. Binding of 1251. mm at 37CC and In 2 h at 4C. Scatchard approximately 6000 receptor sites per cell
and a K. of 1.37 nM. Binding data at 37CC indicated a rapid internalization of rTNF-a, Following this receptor-mediated interaction, recombinant TNF-a was found to inhibit the migration of PMN5 under agarose and to enhance PMN production of superoxide anion (O) in a dose-dependent manner. Furthermore, rTNF-ct-activated PMNs caused a marked disruption of human umbilicalvein-derlved endothelial cell monolayers and caused Inhibition of their proliferative activities. These data substantiate the role of TNFa as an activator of PMN functions and indicate that PMN/TNF-a/endothelial cell interactions may play a major role in Inflammatory reactions. Key words:
migration
inhibition,
cytokines,
INTRODUCTION Human exert mor
polymorphonuclear
antibody-dependent cells, virus
neutrophils
cytotoxic infected cells,
(PMNs)
can
activities against tuand other targets
114,16,35]. Activated PMNs from normal donors are known to produce oxygen-derived metabolic products which may cause target-cell destruction [111 and inactivation of virus [20]. The involvement on PMN in inflammatory
responses. tumor surveillance, and immune mechanisms has been suggested [7,9,21]. Studies recently conducted in this laboratory have demonstrated that recombinant human tumor necrosis factoralpha (rTNF-a) is a potent stimulator of PMN-mediated antibody-dependent cellular cytotoxicity [33]. More recent studies have provided evidence that rTNF-a stimulates PMN production of H2O2 [23] and PMN adherence to umbilical vein endothelium in vitro [15]. The finding
superoxide
anion,
endothelial
cells
The results demonstrate that rTNF-a binds to PMN via a single class of specific, high-affinity receptors and that it inhibits the migration of PMNs and also activates them to produce significant quantities of (Os). Moreover, rTNF-cs-activated PMNs inflicted a marked disruption of HEC monolayers and inhibited the proliferative activities of endothelial cells. These findings emphasize the importance of TNF-a/PMN/endothelial cell interaction in inflammatory reactions.
regulatory
that rTNF-a can stimulate interest since PMNs have cell injury 118,32] during greater
interest
perhaps
PMN functions been implicated inflammatory is the
demonstration
is of particular in endothelial responses. Of that
rTNF-
a has modulatory effects on endothelial cells in vitro [10,26,36] The purpose of this study was to characterize the binding of rTNF-a to human PMNs and to examine the results of this interaction on PMN functions in vitro including migration under agarose, production of superoxide anion, (O) and effects of human endothelial cell (HEC) monolayers. 1987
Alan
R. Liss,
Inc.
MATERIALS
AND METHODS
Recombinant
Human
Factor-Alpha
(rTNF-a)
Tumor
Necrosis
The cloning of the cDNA for human TNF-cu and its expression in E. coli have been reported [28]. Recombinant TNF-a used in this study was purified to greater than 98% purity (Dr. E. Rinderknecht, Genentech, Inc.) and showed structural characteristics identical to those described for the natural molecules [I]. Titers of rTNFa were calculated on the basis of its cytotoxic actinomycin-D-treated L929 mouse fibroblast and showed a specific activity of 7-8 X i0
Received
July
18. 1986:
accepted
September
25.
activity on cells [2] U/mg pro-
1986.
Reprint requests: M.R. Shalaby. Department of Pharmacological ences. Genentech. Inc.. 460 Point San Bruno Boulevard, South Francisco. CA 94080.
SciSan
Activation tein. Concentrated rTNF-a preparations were tested for the presence of contaminating bacterial endotoxin by using the limulus ameobocyte lysate (LAL) gel clotting method (Mallinkrodt, St Louis, MO). Results of LAL showed removal of endotoxin to levels which do not affect PMN functions [33]. Chemicals,
Media,
and
Reagents
Cytochrome c. phorbol-l2-myristate 13-acetate (PMA), and superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St Louis, MO). Minimal essential medium (MEM), RPM! 1640, L-glutamine, non-essential amino acids, Hank’s balanced salt solution (HBSS), Earle’s balanced salt solution (EBSS), and pencillin/
previously 1640 with supplement icillin + fluency in at 37#{176}C in interference
of Polymorphonuclear
Neutrophils
197
described 1391- Cells were cultured in RPMI 10% HS. 15% FBS. endothelial cell growth (150 ug/mL), and antibiotics (80 U/mL pen80 gug/mL streptomycin) and grown to contissue cluture flasks (T-25 cm2) by incubation 5% CO2 humidified atmosphere. To eliminate by contaminating blood cells, these primary
cultures were subjected to a brief trypsinization, washed, then suspended in media plus 10% HS, cultivated at uniform density in 96-well (2 x l0 cells/culture) or 24well (5 x l0 cells/culture) plastic plates and allowed 24-48 before
h of use.
incubation
to
form
confluent
monolayers
Receptor Binding Studies streptomycin were purchased from Grand Island Biological Co. (GIBCO. Grand Island, NY). Fetal bovine serum Recombinant TNF-a was labeled by the iodogen method [13] as described [3]. This resulted in biologi(FBS) (Hyclone, Logan. UT) and pooled human AB cally active ‘251-rTNF-a with a specific activity of - 804 serum (HS) (Peninsula Memorial Blood Bank, South San Francisco, CA) were heat inactivated for 30 mm at 56#{176}C Ci/mmol protein. Assuming monoiodination by this prior to use. method, approximately 50% of the rTNF-a molecules Sodium chromate, Na,5tCrO4 (1 mCi/mL), and 3Hwere labeled at tyrosine residue 87 as revealed by amino acid sequence analysis of this peptide. Further analyses thymidine (40-60 Ci/mmol) were purchased from Amerwere performed as detailed previously [3]. The results sham (Arlington Heights, IL). Affinity purified rabbit showed that the labeled protein maintained the structural antibodies against human rTNF-a (Ab) were provided by and biologic properties identical to those of the unlabeled Dr. K. Bechtol. Hybridoma Research. Genentech, Inc. protein.
Separation
of PMNs
Heparinized syringes were used for collection of blood from healthy donors. Heparinized blood mixed with an equal volume of saline was layered onto Ficoll-Hypaque gradients (sp. gr. 1.08). centrifuged at 400g for 40 mitt at room temperature. Mononuclear cells isolated at the plasma-Ficoll interface were removed and used for preparation of plastic-nonadherent (lymphocyte-enriched) and plastic adherent (monocyte-enriched) cell populations as described [5]. The two populations contained 2% or less PMNs, as determined by Giemsa-stained cytopreparations. The PMN-rich pellet was washed in saline and subjected to dextran sedimentation followed by hypotonic lysis for removal of residual red blood cells. Polymorphonuclear neutrophils were then washed in EBSS and suspended in MEM supplemented with 10% FBS for use in receptor-binding experiments, or in HBSS with 2% HS for use in the migration-inhibition assay. or in EBSS for use in the cytochrome-c-reduction assay. or in RPM! 1640 with 10% HS, L-glutamine, and non-essential amino acids for use in endothelial cell experiments.
Preparation
of Human
Endothelial
Cells
Human umbilical cords were obtained from Dr. Dennis K. Fujii of the Department of Obstetrics and Gynecology and Reproductive Science, University of California, San Francisco. For isolation of umbilical cord veins were subjected to collagenase treatment (1 mg/mL media) as
For receptor-binding experiments, aliquots of freshly isolated PMNs (2 x 106 cells/mL medium) in 12 x 75 mm tubes with 0.2 x 106 cpm of 251-rTNF-a were incubated in the presence or absence of increasing concentrations of unlabeled rTNF-a. After various times of incubation at 37#{176}C or 4#{176}C, the cells were washed three times in media and cell-bound radioactivity was determined in a gamma counter. Nonspecific binding (cellbound radioactivity as determined in the presence of 100 mM unlabeled rTNF-a which is 1000-fold in excess of the 25I-rTNF-a used) was 10% of the total binding. The data are presented as the mean specific binding (cpm) (total binding non specific binding). Migration
Inhibition
Assay
Agarose gel poured into plastic plates (Falcon 3002) was used as described [6]. Briefly, 8-12 x PMNs in 50 uI of HBSS with 2% HS were incubated the absence (control) or presence of rTNF-a (100 mL), rTNF-a plus antibodies, or antibodies alone for
No. l0’ in U/ 30
mm at room temperature. Two millimeter wells were punched in the agarose and 5 uL of control or test PMNs were dispensed in each of triplicate wells. Following 18-20 h of incubation, cells were fixed with 5% glutaraldehyde, agarose was removed, and cells were stained with Giemsa. For each migration area, two random diameters were measured using a dissecting binocular microscope fitted with an eyepiece micrometer. The net
198
Shalaby
et al
area of migration was calculated of the central well. The SEM
by subtracting of triplicate
the area areas was
consistently < 2% of the mean data are expressed as migration calculated as follows.
net migration index (MI),
area. which
=
Mean
net
MI area of rTNF-a
migration
Mean
net
migration
treated
of control
The was
incubated for 3 d and then labeled during the last 6 h of incubation with 2 j.cCi/well 3H-thymidine. Using an automated sample harvester, the cells were harvested onto glass-fiber discs, the discs were allowed to dry, and 3Hthymidine incorporation was determined using a Beckman scintillation spectrometer (Model LS860). Data are presented as mean counts per minute ± SE.
PMNs
PMNs
Statistical Paired
Analysis design
Student’s
t-test
was
used
for the analysis
of data.
Measurement
of Superoxide
Anion
(Ofl
Superoxide anion production by PMNs was measured by the cytochrome-c-reduction assay as detailed previously [30]. Briefly. PMNs suspended in EBSS were dispensed in flat bottom microtiter plates (Falcon No. 3075) at 5 X I0 cells/culture. Various concentrations of rTNFa or other stimuli prepared in EBSS were added to replicate cultures (eight per group). Aliquots of cytochrome c in EBSS were then added (0.2 mg/culture), and the plates were incubated at CO, incubator. Thereafter, (OD) were measured at various using an automated enzyme with a 550 nM filter. The
37#{176}Cin a humidified 5% changes in optical density time points of incubation immunoassay reader fitted stimulus PMA, known to stimulate oxidative metabolism of PMNs [22]. was added to replicate cultures (25 ng/culture) as a positive control. In each individual case, additional control cultures were performed to demonstrate the inhibition of cytochromec-reduction in the presence of SOD (30 U/culture). Data are expressed as nmole 02 production per 5 x 10” PMN and were calculated as follows: (Mean 0.0. readings/ 6.3) x 100130]. PMN
Effects
on HEC
RESULTS -
Binding
E
ft
Monolayers C
In order were able HEC with
to determine whether rTNF-a-activated PMNs to affect the structure of HEC monolayers,
were established untreated PMNs
in 24-well or PMNs
plates and coincubated pretreated for 2 h with
rTNF-a. The PMNs were washed prior to addition to HEC cultures. After incubation for 24 h, culture media were removed and the monolayers were stained with crystal violet and subjected to microscopic examination. The influence of rTNF-a-activated PMNs on the proliferative activities of endothelial cells was examined in additional experiments. Thus, HEC monolayers established in 96-well microtiter plates were used as follows. After removal of culture fluids, groups of quadruplicate cultures received either media only, media with PMNs (0.5 x 106 cells/culture, 10: 1 ratio), or media with PMNs plus rTNF-a. Additional controls included HEC monolayers incubated in the presence of rTNF-a only and cultures of PMNs with and without addition of rTNFa in wells without endothelial cells. The final volume in each of these cultures was 0.2 mL. The cultures were
Studies
We examined the specific binding of ‘25I-rTNF-cu to various blood cell populations by performing three independent experiments. Representative results (Fig. I) demonstrate that PMNs exhibited greater specific binding than populations of lymphocyte-enriched, monocyte-enriched, or red blood cells and that PMNs, monocytes, and lymphocytes are all major targets for TNF-a binding. Results of a similar experiment performed in triplicate showed a mean specific binding of 2385, 1469, 801, and 141 cpm for PMNs, lymphocyte-enriched, monocyte-enriched, and red blood cells, respectively. The differences in binding to PMNs versus other cells at 4#{176}C
C
PMN Lymphocyte Enriched Monocyte Enriched
0 0
a. (I)
Red Blood Cells
Fig. 1. Receptor binding of 125l-rTNF-a to various blood cells at 4u(, Aliquots (2 x iOt cells/mL medium) of either PMNs, lymphocyte-enriched, monocyte-enriched, or red blood cells with 0.2 x 106 cpm of 1251-rTNF-a were incubated in the sb. sence or presence of 200 nM rTNF-a for 3 1/2 h. The cells were washed three times and cell-bound radioactivity was deter. mined. Nonspecific binding was 10% of total binding. Data are presented as mean specific binding (cpm) (total bInding nonspecific bindIng). Results of a similar experiment performed at 37CC showed specific binding (cpm) of 1143 for PMNs which is 30-50% greater than specific binding for lymphocyte-enriched or monocyte-ennched populations.
Activation
of Polymorphonuclear
Neutrophils
199
iment, performed in triplicates at 4#{176}C and 37#{176}C,showed that only 1-5% of the total radioactivity could be detected in precipitates from supernatants of PMN. lymphocyte5000enriched, monocyte-enriched, or red blood cell populations. Rather than the shedding of ligand-receptor complex. these results and those presented in Fig. 2 suggest 4000possible internalization of the molecule, especially at 37#{176}C. Because rTNF-/3 (also called lymphotoxin) and recombinant gamma interferon (rIFN-’y) can also activate PMN 3000functions 133], it was of interest to determine whether these lymphokines interact with PMN through common receptors. Three independent experiments were per20000 -formed and representative result, shown in Fig. 3, indicate that binding of the labeled rTNF-a can be displaced by unlabeled rTNF-a and rTNF-3, but not by rIFN-y. 20 BC 240 These results suggest that, similar to other cell types 13], PMNs have TNF receptors which can be occupied by Tme (m:nutes) both TNF-a and TNF-13, whereas IFN-’y has distinct Fig. 2. Time course of ‘25l-rTNF-a binding to PMNs at 37#{176}C (#{149})receptors. and 4#{176}C (s). Aliquots of PMN5 (2 x 106 cells/mL medium) In The competition binding of ‘251-rTNF-a with unla12 x 75 mm tubes with 0.2 x 106 cpm ‘25i-rTNF-a were Incubeled rTNF-a to PMN at 4#{176}C was examined (Fig. 4). bated in the absence or presence of 200 nM rTNF-o. After The Scatchard analysis (inset) of the completion curve indicated times of incubation the cells were washed three times indicates that PMNs have a single class of high-affinity In media and cell-bound radioactivity was determined. Nonspecific binding was 10% of total binding, Each data point receptors with a K1, of 1.37 nM and approximately 6000 represents the mean of duplicate samples and data are plotted receptor sites per cell. The competition data at 37#{176}C as specific binding after various times of incubation. revealed a similar affinity constant but only 600 receptor
I
were significant by analysis of variance for multiple group comparison (p < 0.01). The binding of rTNF-a to PMNs was studied in more detail since they displayed a greater number of rTNF-a receptors compared to other populations tested. The kinetics of binding of 25I-rTNF-a to PMN at 4#{176}C and at 37#{176}C (Fig. 2) indicate that the binding reaches maximum levels within 30 mm at 37#{176}C and then declines. At 4#{176}C, it takes 2-4 h for the binding to reach a plateau. with maximum values two-fold greater than those obtained at 37#{176}C.In addition, a longer incubation time at 4#{176}C did not result in the decreased binding which was observed at 37#{176}C (Fig. 2). That the observed decline in binding at 37#{176}C was not simply due to formation of ligand-receptor complexes shedded in the medium was tested as follows. Various cell populations used in a repeat receptor-binding assay were pelleted by centrifugation and the supernatants were removed and tested for the presence of soluble receptors. Supernatants were treated with polyethylene glycol and bovine gamma globulin for 10 mm at room temperature; the resulting precipitates were isolated by centrifugation and their radioactivity was determined. These conditions have been found to precipitate soluble rTNF-a receptors (Dr. R. Aiyer, Genetech, Inc.). The results of this exper-
2500T
. O
2000-
500#{176}0
#{246}000 a) (I)
500-
contrd
nfl
rTNFtt
rTNFI3
rlFN-7
Fig. 3. Competition of rTNF-a, rTNF-d, and rIFN-5 with 1251. rTNF-cu for binding to PMNs at 4#{176}C. Aliquots of PMNs (2 x 106 cells/mL medium) in 12 x 75 mm tubes with 0.2 x i06 cpm ‘25TNF were incubated in the absence (control) or presence of 200 nM of either rTNF-a, rTNF-f or rIFN-y, After 3 1/2 h of incubation cells were processed for counting and data are presented as described in legend to Fig. 2. SE v 7%.
200
Shalaby
et al sites
per
cell,
observed
6
i.e.,
Inhibition of PMN
I 5
one-tenth
at 4#{176}C (not
the
total
receptor
number
shown). Migration
Results of seven different experiments, illustrated in Fig. 5, demonstrate that rTNF-a (100 U/mL) is effective in inhibiting the migration of PMNs on a plastic surface.
40O0
This effect was not due to cytotoxicity since control and treated PMNs showed equal trypan blue viability counts (85% ± 2%). The effect of rTNF-a was reversed by the addition of specific antibodies. Treatment of cells with antibodies alone had little or no effect on PMN migration
3000’-
a)
2000
(Fig.
5).
Superoxide We examined
rTNFcx
nM)
Fig. 4. Competition curve of 125I-rTNF’a with unlabelled rTNFat 4#{176}C. Aliquots of PMN5 (2 x 106/mL medium) in 12 x 75 mm tubes with 0.2 x 106 cpm ‘251-rTNF-a were incubated in the absence or presence of indicated amounts of unlabeled rTNF-a. After 3 1/2 h of incubation, cells were processed for counting and data are presented as described in legend to Fig. 2. A Scatchard plot of data is shown (inset).
Radical the
Production influence
of rTNF-a
on O
produc-
tion measured at different time points during a 3 h incubation at 37#{176}Cusing cells from at least four different donors. The results of a representative experiment illustrated in Fig. 6 (panel A). show that PMNs incubated in the presence of different doses of rTNF-a produced significant levels of SOD-inhibitable O in a dose- and timedependent manner (p < 0.01 as revealed by statistical analysis of data from four similar experiments). Activation by PMA (Fig. 6, panel B) resulted in levels of SODinhibitable O that were significantly greater than those caused by rTNFa (p < 0.01). Interestingly, the presence of both PMA and rTNF-a (200 U/mL) in PMN cultures resulted in the production of additive quantities of O (Fig. 6, panel B). Moreover, rTNF-a stimulation of O production reached its peak after 2-3 h of incubation compared to only 1 h for PMA.
x a)
Data number
from additional experiments of donors are summarized
TABLE Human
1. rTNF-a-Activation PMN5
with PMNs from a in Table I. The extent
‘D
C C
0
a Culture conditions
Dl
rTNF
rTNFcx + Ab
Ab
Fig. 5. inhibitIon of PMN migration by rTNF-a. Migration of PMN5 under agarose following incubation at room temperature for 30 mm In the absence or presence of either rTNF-a (100 UI mL), similar preparation of rTNF-a neutralized with Ab or with Ab alone. Each data point represents the mean MI of triplicate determination, SE v 2%. Results of seven different experi. ments are shown.
of 01
Nanomoles
Production
00
Cumulative
productio
by
n/S
daub
x t0
PMNs#{176}
p vatuesc
Medium
0.76
± 0.13
(tO)
PMA
5.08
± 0.23
(9)
