Bioscience Reports, Vol. 7, No. 11, 1987
Protein Synthesis is Activated in Primed
Neutrophils" a Possible Role in Inflammation
Valerie Hughes, John M. Humphreys and Steven W. Edwards 1
Received November 18, 1987
KEY WORDS: neutrophils; protein synthesis; chemotactic peptide; respiratory burst; inflammatory
response.
ABBREVIATIONS: luminol, 5-amino-2,3-dihydrophthalazine-l,4-dione; TCA, trichloroacetic acid; 2DPAGE, two-dimensional polyacrylamide gel electrophoresis; fMet-Leu-Phe, N-formyl-L-methionyl-Lleucyl-L-phenylalanine; DMSO, dimethyl sulphoxide; SDS, sodium dodecyl sulphate.
Circulating human neutrophils exhibited low rates of protein biosynthesis, as
determined by their ability to incorporate [35S]methionine into TCA-precipitable
material. Exposure of cells to the chemotactic peptide (N-formyl-L-methionyl-Lleucyl-L-phenylalanine) increased their rate of protein synthesis, and the maximal
stimulation of biosynthesis by this inflammatory agent was observed at 0.1 pM: this
concentration of chemotactic peptide "primed" neutrophil activity and only activated
the oxidase of these cells by 8 % of maximum. The newly-synthesized proteins were
analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with
those synthesized in control cells. Two classes of proteins were observed in "primed"
cells. The first of these comprised proteins whose rate of biosynthesis changed very
little upon "priming" whereas the second class comprised proteins whose rate of
synthesis increased greatly after exposure to chemotactic peptide. The fMet-Leu-Phe
stimulated protein synthesis was inhibited by actinomycin D and cycloheximide
showing that this phenomenon required both transcription and translation. We
propose that these fMet-Leu-Phe regulated proteins play an important role in the
function of neutrophils during an inflammatory response.
INTRODUCTION
Polymorphonuclear leukocytes are phagocytic cells of the immune system which are
highly specialized for their crucial role of phagocytosing and killing yeasts and bacteria
during infections. Thus, they possess a battery of cytotoxic enzymes and associated
Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, UK.
i To whom correspondence should be addressed.
881
0144-8463/87/1100-0881505.00/0 9 1987PlenumPublishingCorporation
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Hughes, Humphreysand Edwards
pathways which can be utilized for cell killing, and these can be activated by a number
of soluble or particulate stimuli (Klebanoff and Clark, 1978; Karnovsky and Bolis,
1982). Much work over recent years has focussed on identifying these pathways (Segal
and Jones, 1978; Rossi, 1986), and determining how they may be activated and
regulated under pathological conditions (Edwards et al., 1984; Edwards et al., 1987a).
These cytotoxic processes may be broadly divided into O2-independent (Elsbach and
Weiss, 1983; Spitznagel, 1984) and O2-dependent mechanisms (Fantone and Ward,
1982; Babior, 1984), and much debate exists in the literature as to the relative
importance of these processes in microbial killing, although the relative efficiency of
any one mechanism depends, at least in part, upon the biochemical properties of the
target microorganism (Edwards et al., 1987b).
Whilst it is convenient and usual to measure neutrophil activity in vitro under
conditions favouring maximal responsiveness, more recent approaches have utilized
experimental designs approximating more closely in vivo conditions, thus offering the
possibility that we can understand more clearly the function and activity ofneutrophils
during an acute inflammatory response. Of particular interest is the responses of
neutrophils to concentration gradients of inflammatory mediators since in vivo they
must respond to such gradients as they are signalled to migrate from the bloodstream
and move towards the inflammatory site. It has thus been shown that neutrophils can
be "primed" in vitro by low concentrations of inflammatory agents (usually
concentrations 10-fold lower than those necessary to evoke activation per se) and this
process renders neutrophils more responsive towards exposure to a second stimulus
(McCall et aI., 1979; Van Epps and Garcia, 1980; English et al., 1981; Bender et al.,
1983; McPhail et al., 1984; Dewar and Baggiolini, 1985). The molecular mechanisms
underlying "priming" and enhanced responsiveness are largely unknown.
Based largely on morphological evidence indicating a relative scarcity of
ribosomes and endoplasmic reticulum, it is assumed that mature, bloodstream
neutrophils have little, if any, capacity for protein synthesis (Klebanoff and Clark,
1978). Indeed, since many neutrophil functions such as phagocytosis and reactive
oxidant generation can occur in vitro in the presence of inhibitors of RNA and protein
synthesis (Cline, 1966), macromolecular biosynthesis is not even considered necessary
for efficient microbial killing during infections. However, we have re-examined the
capacity of neutrophils for protein synthesis in order to understand more clearly the
molecular mechanisms which occur during "priming" to render the neutrophil more
functionally active. We show, for the first time, that "priming" is associated with a 5-6
fold increase in the rate of protein synthesis and propose that the newly-synthesized
proteins play an important role in the acute inflammatory response.
METHODS AND MATERIALS
Preparation of Neutrophils
Human Polymorphonuclear leukocytes (neutrophils) were isolated from
heparinized venous blood from healthy volunteers utilising either a dextran/ficoll
sedimentation procedure (Edwards and Swan, 1986) or Mono-Poly Resolving
Medium (Flow Laboratories) as described in the manufacturer's instructions. After
Protein Synthesisin Neutrophils
883
purification, cells were suspended either in a buffer containing (mM): NaC1, 120: KC1,
4.8; KH/PO4, 1.2; CaC12, 1.3; MgSO4, 1.2; hepes, 25 (pH 7.4) and 0.1% bovine serum
albumin, or RPMI 1640 medium (Flow Laboratories) containing 0.5 % foetal calf
serum. Neutrophils ( > 9 8 % purity) were counted using a Fuchs-Rosenthal
haemocytometer slide and used within 4 hr of preparation.
Measurement of [35S]Methionine Incorporation
Neutrophils were suspended in RMPI 1640 medium containing 0.5 % foetal calf
serum to 2-4 x 107 cells/ml at 37~ To each incubation mixture, 60 #Ci/ml (final
conc.) of [3SS]methionine was added and neutrophils were maintained in suspension
by gentle agitation. After a 10 min pre-incubation period, the chemotactic peptide,
fMet-Leu-Phe (dissolved in DMSO) was added (at the stated concentration), whilst
control suspensions contained no additions or the corresponding concentration of
DMSO as used in test suspensions. After suitable time intervals, aliquots were
removed and proteins precipitated with 10% TCA (final conc.) containing 2% (w/v)
casein hydrolysate for 16 hr at 4~ Precipitated proteins were then filtered onto
Whatman GF/C filters, washed six times with 10% TCA and finally once with ethanol.
The filters were then dried, mixed with 4 ml of Scintillation Cocktail T (BDH
Chemicals) and counted using a Packard Scintillation Counter.
Two-Dimensional Polyacrylamide Gel Electrophoresis
Neutrophils were suspended in RPMI 1640 medium containing 0.5 % foetal calf
serum plus 60pCi/ml [3SS]methionine and incubated for 1 hr at 37~ After this
period proteins were precipitated with 10% TCA for 16 hr at 4~ and then centrifuged
at 11,600 g for 5 rain. The supernatants were discarded and the protein precipitates
washed five times with 1 ml aliquots of ether (to remove traces of TCA) and after the
final wash and removal of supernatants, the pellets were warmed to 37~ to remove
residual traces of ether. Protein precipitates were then analysed by two-dimensional gel
electrophoresis employing systems utilizing either isoelectric focusing (IEF)
(O'Farrell, 1975) or non-equilibrium pH gradient electrophoresis (NEPHGE)
(O'Farrell et al., 1977) for the first dimension. The second dimension employed a 13 %
polyacrylamide gel containing SDS. After electrophoresis, gels were soaked in DMSO
for 3 hr (with 3 changes) prior to soaking in a PPO (2.5-diphenyl oxazole) solution in
DMSO (20 % w/w, final conc.). After extensive washing in double-distilled water, gels
were dried and exposed to pre-flashed Fugi RX X-ray film at - 7 0 ~ for 2-3 weeks.
Chemiluminescence Measurements
Suspensions of neutrophils (1-2x 106 cells/ml) were incubated with 10#Mluminol (5-amino-2,3-dihydrophthalazine-1, 4-dione) as described previously
(Edwards, 1987) and chemiluminescence was measured using an LKB Watlac 1250
luminometer.
884
Hughes, Humphreys and Edwards
Chemicals
fMet-Leu-Phe, actinomycin D, cycloheximide and luminol were from Sigma
whereas [ 35S]methionine was from Amersham International. All other chemicals were
of the highest purity available.
RESULTS
Effect of fMet-Leu-Phe Concentration on Protein Synthesis
Exposure of neutrophils to the chemotactic peptide fMet-Leu-Phe stimulates
chemotaxis and activates the respiratory burst to generate a series of reactive oxygen
metabolites. Also, this compound can "prime" the oxidant-generating pathway to
produce enhanced levels of oxidants upon the addition of a second stimulus, but the
concentration required for priming is 10-fold lower than that which illicits a
respiratory burst p e r se (see Introduction). Therefore, we examined the effect of fMetLeu-Phe concentration on the rate of [35S]methionine incorporation into TCAprecipitable proteins. As shown in Fig. 1, at 2 pM-fMet-Leu-Phe the rate of protein
synthesis was not enhanced above the low rate observed in the absence of this
compound, but as the concentration was decreased, so the rate of synthesis
correspondingly increased. Thus, at 0.1#M-fMet-Leu-Phe the rate of protein
synthesis was stimulated five-fold above control values. Thus, concentrations of fMetLeu-Phe which "prime" the oxidase to produce enhanced levels of oxidants upon
A
o')
I
o
1'--
2
0
0
I
I
I
I
0.5
1
1.5
2
FMLP
concn.
(I~M)
Fig. 1. Effect of fMet-Leu-Phe concentration on neutrophil protein
synthesis. Neutrophils (2 x 107/ml) were suspended in RPMI medium
containing 0.5 % foetal calf serum and 60 #Ci/ml [35S]methionine at
37~ as described in Methods and Materials. After a 10min preincubation period, fMet-Leu-Phe was added to each tube at the
concentration indicated and after 60 min incubation, the radioactivity
incorporated into TCA-precipitable material was measured. Counts
presented are corrected for those obtained in control suspensions whose
rate of incorporation was 1400cpm.
Protein Synthesis in Neutrophils
E
885
60
o
re.)
40
e.-
20
u
Q)
t-
/"
O
4
TIME
(min)
Fig. 2. Effect offMet-Leu-Phe concentration on neutrophil
oxidase activity. Suspensions of neutrophils were incubated
with 10#M luminol, stimulated with fMet-Leu-Phe (a,
1/~M; b, 0.1 gM, final cones) and the chemiluminescence
response measured.
s u b s e q u e n t s t i m u l a t i o n , also s t i m u l a t e d a n i n c r e a s e d r a t e o f p r o t e i n synthesis in
n e u t r o p h i l s . F i g u r e 2 s h o w s t h a t 0.1 g M - f M e t - L e u - P h e s t i m u l a t e d o n l y 8 ~ of t h e
o x i d a s e a c t i v i t y s t i m u l a t e d by 1 g M - f M e t - L e u - P h e ,
as m e a s u r e d b y l u m i n o l dependent chemiluminescence.
6co
i
ov -
4
X
:E
2
o
0
I
I
I
I
30
60
90
120
TIME
(rain)
Fig. 3. Time course of fMet-Leu-Phe stimulated protein synthesis.
Neutrophil suspensions (6 x 106 cells/ml) were incubated as described in
the legend to Fig. 1. At time zero, 0.1 ~tM fMet-Leu-Phe (final cone.) was
added to the test suspension (final DMSO cone. of 0.1 ~), while control
suspensions contained no additions or 0.1 ~o DMSO only. At time
intervals portions were removed from the test and control suspensions,
and proteins were precipitated as described in Materials and Methods.
Data presented for the stimulated suspension have been corrected for
control values which ranged from 100-2700 cpm. Typical results from
at least 5 separate experiments.
886
Hughes, Humphreysand Edwards
Time Course of fMet-Leu-Phe Stimulated Protein Synthesis
The time course of fMet-Leu-Phe stimulated protein synthesis was then
determined by removing aliquots from suspensions of neutrophils incubated in RPMI
1640 medium (supplemented with 0.5 % foetal calf serum) in the presence or absence of
0.1/~M-fMet-Leu-Phe and measuring the incorporation of [35S]methionine into
TCA-precipitable material. Figure 3 shows that the rate of fMet-Leu-Phe stimulated
protein synthesis increased steadily for the initial 60 rain after the addition of stimulus.
After this period, the rate rapidly declined and was undetectable above the
unstimulated rate by 2 h after the addition of fMet-Leu-Phe (presented data are
corrected for the rate of protein synthesis in control, unstimulated suspensions
incubated under identical conditions).
Separation of Newly-Synthesized Proteins by 2D-PAGE
It was then necessary to determine whether this fMet-Leu-Phe dependent
increased rate of protein synthesis represented either:
(a) an increased rate of synthesis of all proteins which are normally synthesized at
low rates in mature cells or
(b) the increased rate of biosynthesis of a selective group of proteins.
This was achieved by analysis of newly-synthesized proteins by two-dimensional gel
electrophoresis, utilizing for the first dimension either isoelectric focusing (IEF) to
separate acidic/neutral proteins or non-equilibrium pH gradient electrophoresis
(NEPHGE) to separate basic proteins. Polypeptides labelled after incubation for 1 hr
in the presence of 0.1/~M-fMet-Leu-Phe were compared with those labelled in control
cell suspensions incubated under identical conditions in the absence of fMet-Leu-Phe.
Separation of [35S]methionine labelled polypeptides utilizing IEF for the first
dimension resolved a number of components (Fig. 4a) which were labelled in control
suspensions. However, in fMet-Leu-Phe treated cells, a much greater number of
labelled polypeptides were resolved (Fig. 4b). Two major groups of polypeptides were
distinguished in fMet-Leu-Phe treated cells as those whose rate of synthesis either (a)
changed very little or (b) increased greatly after fMet-Leu-Phe exposure. When
N E P H G E was used for the first dimension, it was apparent that fMet-Leu-Phe
treatment resulted in a greatly increased rate of synthesis of a large number of basic
proteins which were normally expressed at low rates in untreated cells (Fig. 4c,d).
Effects of Inhibitors of Transcription and Translation
In order to determine whether the increased rate of incorporation of
[35S]methionine into TCA-precipitable material represented increased transcriptional
or translational activity, the effects of actinomycin D and cycloheximide, respectively
were examined. Suspensions of neutrophils were incubated in the presence of 0.1 #M
fMet-Leu-Phe in the absence of presence of 5 #g/ml actinomycin D or 10/~g/ml
cycloheximide. After 1 hr incubation, proteins were precipitated and the incorporation
of [3~S]methionine was measured. Actinomycin D inhibited the fMet-Leu-Phe
Protein Synthesis in Neutrophils
887
Fig. 4. Separation of newly-synthesised proteins by two-dimensional gel
electrophoresis. Both control and fMet-Leu-Phe treated suspensions (0.1 #M final
conc.) were incubated as described in the legend to Fig. 1. After 60 min incubation,
proteins were precipitated with TCA and then analysed by two-dimensional gel
electrophoresis, utilizing either iso-electric focusing (IEF) or non-equilibrium pH
gradient electrophoresis (NEPHGE) for the first dimension. The second dimension
employed a 13% polyacrylamide gel (containing SDS). After electrophoresis,
[aSS]methionine labelled polypeptides were visualized by fluorography. Molecular
weights were determined from suitable protein markers.
activated protein synthesis by over 60%, while cycloheximide reduced the rate of
i n c o r p o r a t i o n to b a c k g r o u n d levels. These data thus show that both transcriptional
and translational activity are required for the increased rate of labelling of proteins.
Protein Synthesis by Contaminating Monocytes
It is generally appreciated that circulating monocytes have a considerable
capacity for protein synthesis and therefore it was necessary to eliminate the possibility
that c o n t a m i n a t i n g m o n o c y t e s contributed to the overall rate of protein synthesis in
the suspensions of neutrophils. This was achieved by (a) estimating the extent of
888
Hughes, Humphreysand Edwards
monocyte contamination in the neutrophil suspensions and (b) determining the rate of
protein synthesis attributable to contaminating monocytes during incubation under
these conditions. Neutrophils were found to typically contain about 2%
contamination by monocytes, as determined by Wright's staining. Therefore, the rate
of incorporation of [35S]methionine was measured in suspensions of neutrophils
c o n t a i n i n g 10 7 cells/ml and also in suspensions containing 2x 105 and 5 x 105
monocytes/ml (representing 2 and 5 % contamination by monocytes, respectively).
Suspensions were then incubated for 1 hr in the presence or absence of 0.1 #M fMetLeu-Phe and the rate of labelling measured. In all monocyte suspensions, the rate of
incorporation of [35S]methionine was lower than that of the untreated neutrophil
suspensions. These experiments thus show that the enhanced rate of protein synthesis
in fMet-Leu-Phe treated suspensions of neutrophils is not due to a contribution from
contaminating monocytes.
DISCUSSION
The subject of protein synthesis in mature neutrophils, unlike phagocytosis and
oxidative metabolism, has received surprisingly little attention since it is assumed that
circulating, bloodstream cells are terminally differentiated and that protein synthesis is
restricted to immature, precursor cells. Indeed, since it is also assumed that mature
neutrophils have a very short life-span both in vivo and in vitro, little attention has
focussed on the longer-term effects of inflammatory agents on these cells, i.e. time
scales beyond the duration of the respiratory burst. Only a few reports have directly
measured rates of protein synthesis in these cells (Granelli-Piperno et al., 1979;
Blowers et al., 1985) but more recently, a role for protein synthesis in mature cells has
been proposed since it has been reported that the effects of immune interferon on the
modulation of some neutrophil functions can be prevented by inhibitors of protein
synthesis (Berton et al., 1986; Steinbeck et al., 1986). We show here, for the first time,
that protein synthesis is stimulated by exposure of neutrophils to a chemotactic agent
and that the concentration of fMet-Leu-Phe required for maximal stimulation of
protein synthesis (0.1 #M, Fig. 1) only stimulated the respiratory burst (as determined
by luminol-dependent chemiluminescence) by 8 % (Fig. 2). This concentration offMetLeu-Phe is similar to the optimum concentration required for "priming" of the
NADPH oxidase to produce enhanced levels of oxidants upon subsequent exposure to
stimulus.
This "priming" of neutrophils in vitro by low concentrations of chemotactic
factors has been proposed to mimic a physiological phenomenon which enhances their
responsiveness as they pass through a concentration gradient of inflammatory
mediators during an inflammatory response. Thus, upon arrival at a site of infection,
their enhanced cytotoxicity ensures more efficient microbial killing. We now show here
that such "priming" of neutrophils is also associated with a marked stimulation of
protein synthesis. Resolution of proteins by two-dimensional gel electrophoresis
revealed that two classes of proteins could be identified: those whose rate of labelling
changed very little and those whose rate of labelling increased greatly after exposure to
fMet-Leu-Phe. Since this increase in incorporation of [ aSS]methionine was reduced by
Protein Synthesis in Neutrophils
889
actinomycin D and cycloheximide, this latter group of proteins represents those whose
increased expression requires both transcription and translation during "priming".
We now propose that these fMet-Leu-Phe regulated proteins in "primed" cells play an
important role in their function during the acute inflammatory response.
It has recently been shown that while mature, circulating bloodstream neutrophils
contain large quantities of fibrinonectin, they actively synthesize little, if any, of this
protein (La Fleur et al., 1987; Marino et al., 1987). However, a marked increase in the
level of m R N A for this protein was observed in neutrophils isolated from the synovial
fluid of patients with inflammatory diseases such as rheumatoid arthritis (La Fleur et
al., 1987) and this was reflected in an increased rate o f de novo synthesis of this protein
(plus a few other proteins) in such cells (Beaulieu et al., 1987). Thus, these observations
support our data presented here, since it is assumed that in inflammatory disorders
such as rheumatoid arthritis, neutrophils have been "primed" and activated in vivo.
Marino et al. (1987), using a fibrinonectin gene probe, confirmed that this protein is
not actively synthesized in circulating neutrophils, but in contrast to the above
findings, did not observe increased levels of transcripts when the cells were stimulated
in vitro. However, in these experiments, stimulation was achieved by incubating the
neutrophils with 10 # M fMet-Leu-Phe and we have shown that stimulated protein
synthesis is undetectable at concentrations of this agent used in excess of 1/~M (Fig. 1).
Further work is now necessary to identify these fMet-Leu-Phe-regulated proteins
and to determine whether a similar phenomenon occurs in neutrophils which have
been "primed" in vitro. As a first step towards this aim we have constructed a c D N A
library from m R N A isolated from "primed" cells. This work thus dispels the belief that
protein synthesis plays no part in the crucial defensive function of these important cells.
ACKNOWLED GEMENTS
We thank The Nuffield Foundation and Arthritis and Rheumatism Council for
financial support.
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