Unité INSERM U167, Institut Pasteur, Lille, France
Correspondence: Dr. Monique Capron, Unité INSERM U167, Institut Pasteur de Lille, 1 rue du Prof. Calmette, BP 245, 59019 Lille Cedex, France. E-mail: monique.capron{at}pasteur-lille.fr
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(IFN-) and
interleukin (IL)-12. Although PMN do not seem to express IL-10 and
IL-13, no information exists on the ability of PMN to produce IL-4.
Therefore intracellular flow cytometry was performed in the presence or
absence of Brefeldin A. Similarly to eosinophils, freshly isolated
neutrophils from normal donors contained low amounts of IL-4, which
significantly increased upon culture with Brefeldin A
(P < 0001). Immunostaining performed on cytospin
preparations of normal granulocytes confirmed the presence of
intracellular IL-4. Using a highly sensitive ELISA, the levels of IL-4
secreted by cultured PMN and peripheral blood mononuclear cells (PBMC)
were compared. PBMC secrete up to 60 times more IL-4 as PMN but, in the
presence of calcium ionophore, only PMN showed a slight but significant
increase in IL-4 secretion (P < 0.05). In conclusion,
we report here the presence within human PMN of intracellular IL-4,
which can at least partly be released under calcium ionophore
stimulation. The relevance of this production of IL-4 by human PMN is
discussed.
Key Words: eosinophils • intracellular flow cytometry • enzyme-linked immunosorbent assay
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(TNF-) and
interleukin (IL)-1ß without omitting the numerous chemokines involved
not only in neutrophil recruitment (IL-8, GRO) but also in tissue
infiltration by monocytes and T cells [monocyte chemotactic protein-1
(MCP-1), macrophage inflammatory protein (MIP)-1 and ß,
IFN--inducible protein-10 (IP-10), MIG, and I-TAC]
[1
2
3
4
5
]. In vitro, the release of most of
these pro-inflammatory cytokines and chemokines by PMN upon stimulation
is increased in the presence of IFN- and inhibited in the presence
of IL-10 [1
].
A type 1 cytokine pattern is classically associated with a
cell-mediated immune response and the activation of phagocytosis by
neutrophils and macrophages. In this context, recent reports suggest
that PMN do also release IFN- [6
] as well as IL-12
[5
, 7
]. In fact, a combination of
lipopolysaccharide (LPS) and interferon- (IFN-) was reported to
induce the synthesis of the IL-12 p40 and p35 subunits, as well as the
secretion of p70, the heterodimeric IL-12 [7
]. In turn,
IL-12 is known to promote a Th1 response by increasing IFN-
secretion by T cells [8
]. Although lymphocytes and
monocyte/macrophages appear to be the main producers of these cytokines
[8
], the overwhelming presence of neutrophils in the
peripheral blood and the fact that their activation leads to a massive
tissue invasion and an enhanced survival, suggest that
neutrophil-derived immunoregulatory cytokines may be of importance in
the inflammatory process.
Clearance of infectious agents by neutrophils and macrophages is closely linked to the resolution of the local inflammation. Therefore, neutrophils are likely to participate in the down-regulation of the inflammatory response by releasing anti-inflammatory mediators like soluble TNF receptors or transforming growth factor ß (TGF-ß) [9 , 10 ]. In the presence of IL-4, the main type 2 cytokine, PMN is also reported to release large amounts of IL-1ra [11 ]. Although IL-10 and IL-13 synthesis has not been detected in PMN [12 ], no information exists on the ability of PMN to produce IL-4 [1 , 13 , 14 ]. Regarding the low levels of IL-4 released in PMN culture supernatants, we rather evaluated the intracellular accumulation of IL-4 in the presence of Brefeldin A. By using intracellular flow cytometry and immunostaining, we were able to demonstrate IL-4 production in neutrophils, ruling out, therefore, a possible contamination by other IL-4-producing cells, like T cells or eosinophils [13 , 14 ].
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Cell purification
Human peripheral blood PMN and peripheral blood mononuclear
cells (PBMC) were isolated from heparinized venous blood of healthy
volunteers by Percoll density gradient centrifugation (d = 1.082
g/L). PBMC were recovered at the interface, whereas the pellet
contained the granulocytes. Erythrocytes were lysed in a cold solution
of 0.15 M NH4Cl, 0.01 M NaHCO3, and 0.01 M
EDTA. The recovered cells were washed and resuspended in
phosphate-buffered saline (PBS). Cell preparations with more than 95%
neutrophils and an average of 2–4% eosinophils were routinely
obtained. Both cell populations were further purified by positive and
negative immunomagnetic selection, respectively, using anti-CD16-coated
microbeads and the MACS system [15
]. The final cell
preparation contained over 99% neutrophils, whereas the eosinophil
population was about 95% pure. All cell populations showed a viability
of 95%, as assessed by trypan blue.
Intracellular flow cytometry
For intracellular staining freshly purified neutrophils and
eosinophils were fixed with 2% paraformaldehyde for 15 min at room
temperature. After washing in PBS, the granulocytes were resuspended in
PBS containing 1% BSA and 0.5% Saponin (permeabilization buffer) for
10 min. The cells were preincubated with 5 µL normal mouse serum for
10 min in order to block nonspecific staining, and further incubated
with PE-conjugated anti-IL-4 monoclonal antibody (5 µg/mL) or
PE-conjugated control isotype (5 µg/mL) for 30 min. The cells were
washed once in permeabilization buffer, once in PBS, and then
resuspended in PBS-0.5% BSA before analysis. Samples were analyzed by
flow cytometry on a Coulter Profil II cytometer (Coultronics, Hialeah,
FL) using the EPICS software. Ten thousands events were usually
acquired per sample. Thresholds were set on control stains.
To assess intracellular IL-4 accumulation, freshly purified granulocytes were cultured in the presence of Brefeldin A (10 µg/mL) for 18 h and cells were then processed for IL-4 staining as described above.
To control the specificity of the intracellular staining in PMN, PE-conjugated anti-IL-4 monoclonal antibodies were preincubated for 15 min with recombinant human IL-4 (10 µg/mL) or with an excess of an irrelevant cytokine (recombinant human GM-CSF at 50 µg/mL) before cell staining.
Immunocytochemistry
Cytospins of freshly purified granulocyte preparations were
fixed in cold acetone/methanol (1:1) for 2 min and, after air drying,
the slides were rehydrated in Tris-buffered saline (TBS) for 10 min.
The alkaline phosphatase anti-alkaline phosphatase (APAAP) method was
used for immunostaining [15
]. After all incubation
steps, cytospins were washed for 3 x 10 min in TBS containing
0.1% BSA. Briefly, after saturation with 3% BSA in TBS for 30 min,
cytospins were incubated with unlabeled anti-human IL-4 or isotype
control monoclonal antibodies at 40 µg/mL in TBS-3% BSA overnight at
4°C. The slides were then incubated with rabbit anti-mouse
immunoglobulins (1:25) in TBS-3% BSA for 1 h at room temperature,
followed by incubation with APAAP complex (1:40) for 1 h. After
washing as before, followed by additional washes for 2 x 10 min
in TBS, the reaction was developed with New Fuchsin substrate (Dako).
The slides were counterstained with Mayer’s hematoxylin and mounted
with Immu-mount (Shandon, Pittsburgh, PA).
Culture conditions
Culture medium consisted of RPMI 1640 supplemented with 10%
heat-inactivated FCS, 2 mM L-glutamine, 100 IU/mL
penicillin, and 100 µg/mL streptomycin. PBMC, PMN, and eosinophils
were then incubated at 37°C, in a humidified atmosphere with 5%
CO2 for 24 h. Freshly purified neutrophils were
cultured for 18 instead of 24 h to avoid apoptosis
[16
]. PBMC and eosinophils were cultured in 24-well
plates, at a concentration of 1 x 106 and 2 x
106 cells/mL, respectively, and neutrophils were cultured
in 12-well plates at a concentration of 5 x 106
cells/mL/well.
For IL-4 accumulation experiments, neutrophils and eosinophils were cultured with Brefeldin A at a final concentration of 10 µg/mL.
For activation experiments, neutrophils and PBMC were cultured in the presence or absence of 1 µg/mL of calcium ionophore (A23187).
IL-4 measurements
To compare the levels of IL-4 released by PMN and PBMC, we used
a very sensitive enzyme-linked immunosorbent assay (ELISA; Diaclone),
according to the manufacturer’s recommended procedure. The levels of
IL-4 released by most cultured PMN in the above conditions were within
the range of the assay (0.2–3 pg/mL), but PBMC samples had to be
diluted 10 times.
Statistical analysis
All results were assessed using the Student’s t
test. Statistical significance was determined with a confidence level
of at least 95%.
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 View larger version (29K): [in a new window] |
Figure 1. Flow cytometry analysis of intracellular IL-4 in cultured neutrophils
(A–D) and eosinophils (E, F). (A) Detection of intracellular IL-4 in
freshly purified PMN (solid line). (B) Detection of intracellular IL-4
in PMN cultured for 18 h in the presence of Brefeldin A (solid
line). Similar results were observed with purified eosinophils (E and
F). Competition assay: although a concentration of 50 µg/mL of
recombinant human GM-CSF proved to be inefficient at blocking the IL-4
antibody (solid line, C), a concentration of 10 µg/mL of recombinant
human IL-4 (solid line, D) was able to totally inhibit the
intracellular fixation of the PE-conjugated IL-4 antibody. Staining
with the isotype-related control Ab is represented by the dotted line
for each experiment.
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Similar results were observed with purified eosinophils (Fig. 1E and 1F) , although the intensity of the staining in the case of eosinophils was less than for neutrophils (Fig. 1E ; MFI = 2.3 ± 1.7, n = 5), indicating that eosinophils from normal healthy donors expressed less IL-4 than neutrophils. Again, when cells were cultured in the presence of Brefeldin A, IL-4 was accumulating within eosinophils (Fig. 1F) . These results suggest that, similarly to eosinophils [17 18 19 20 21 ], neutrophils can produce IL-4. It is interesting that only the 8D4 clone of anti-IL-4 mAb and not the MP4-25D2 clone allowed the detection of intracellular IL-4 by flow cytometry, both in neutrophils and in eosinophils, suggesting some difference in the binding of anti-IL-4 mAb to granulocytes versus lymphocytes.
Kinetic study of intracellular IL-4 accumulation in cultured
neutrophils
A strong accumulation of IL-4 within neutrophils was detected
after culture in the presence of Brefeldin A, suggesting that the weak
IL-4 expression observed on freshly purified cells was due to some IL-4
release from an intracellular store. Therefore we performed a kinetic
study of the intracellular accumulation of IL-4 in the presence of
Brefeldin A at 0, 6, 14, 18, and 22 h. As shown in Figure 2
, a time-dependent accumulation of IL-4 within the cells could be
obtained, with a maximal accumulation being observed between 18 and
22 h. No significant variation in the isotype control peak was
observed (Fig. 2
, dotted lines). These results were indicating that
neutrophils and also eosinophils (Fig. 1F)
could rapidly accumulate
IL-4 and therefore participate actively in the immune response.
 View larger version (24K): [in a new window] |
Figure 2. Kinetic study of intracellular IL-4 accumulation in cultured PMN.
Intracellular detection of IL-4 (solid line) in neutrophils by flow
cytometry after 0, 6, 14, 18, and 22 h of culture in the presence
of Brefeldin A (10 µg/mL). Staining with the isotype-related control
Ab is represented by the dotted line. A representative experiment of
the observed time-dependent accumulation of IL-4 in neutrophils is
shown.
|
 View larger version (89K): [in a new window] |
Figure 3. Immunocytochemical detection of IL-4 in freshly purified granulocytes
from normal donors (96% neutrophils and 4% eosinophils). Cytospin
preparations were incubated with anti-IL-4 mAb (A) or murine IgG1
isotype control antibody (B), followed by detection with the APAAP
method. All neutrophils showed a positive staining for IL-4, and only a
minor proportion of eosinophils (arrow) were weakly positive. No
staining was observed with an irrelevant isotype control. Original
magnification x40.
|
, and
combinations of these with LPS), calcium ionophore proved to be the
most efficient PMN activator to enhance cytokine release, as previously
suggested [16
]. Therefore PMN were cultured in the
absence or in the presence of calcium ionophore (A23187) for 18 h,
and IL-4 was measured in the supernatants through the use of a very
sensitive ELISA assay (<0.2 pg/mL). The spontaneous release of IL-4 by
nonstimulated neutrophils was barely detectable (Fig. 4
). In fact, the amounts of IL-4 measured were so low that most of
them (five out of eight) were below the detection limit of the ELISA.
After stimulation with calcium ionophore, a significant increase in the
release of IL-4 was observed (1.12 ± 1.09 pg/mL compared to
0.25 ± 0.19 pg/mL; P < 0.05).
 View larger version (18K): [in a new window] |
Figure 4. Quantification by ELISA of IL-4 released by cultured PBMC and purified
neutrophils (PMN). The spontaneous release of IL-4 by highly purified
(>99%) nonstimulated neutrophils (n = 8) and PBMC
(n = 4) was compared after 18 and 24 h of culture,
respectively. Although PBMC spontaneously release about 60 times more
IL-4 as PMN (P < 0.005), after calcium ionophore
stimulation (10 µg/mL), only PMN secreted significantly more IL-4
(P < 0.05). The dotted line represents the detection
limit of the ELISA assay: 0.2 pg/mL.
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As already shown by intracellular FACS analysis, freshly isolated
eosinophils, another granulocyte population, seem to contain low levels
of IL-4 [21
]. It is interesting that, although not
significantly, neutrophils presented a slightly higher intracellular
staining for IL-4 than for eosinophils. Furthermore, intracellular FACS
analysis performed with Brefeldin A showed a similar IL-4 accumulation
in neutrophils and eosinophils. It has been hypothesized that the
accumulation of intracellular IL-4 in eosinophils, only in the presence
of Brefeldin A, suggested a continuous release of IL-4 by cultured
eosinophils [21
]. In accordance with this hypothesis,
eosinophils cultured for 24 h with soluble IgA, IgG, or TNF-
all showed a decrease in intracellular IL-4 [20
]. We
suggest that this can also be true for neutrophils.
As demonstrated by Cassatella et al. for IL-12, immunoregulatory cytokines remain undetectable in PMN culture supernatants, unless the cells are stimulated with the right combination of mediators [7 ]. In the present work, neutrophils were stimulated with various combinations of LPS and cytokines or with calcium ionophore to induce the secretion of IL-4 (data not shown). Only calcium ionophore was able to induce IL-4 release by neutrophils, as previously shown for human eosinophils [17 ]. Although IL-4 production by eosinophils has been reported in a growing number of studies [17 18 19 20 21 ], none has detected IL-4 in the supernatant of eosinophils from normal donors (< 3 pg/mL for 106 eosinophils). This suggests that IL-4 released by eosinophils from normal donors do not represent an important source of IL-4. Therefore eosinophils were not included as comparison in the ELISA experiments. The detectable amounts of IL-4 released by cultured neutrophils were so low (about 1 pg/mL) that discriminating between IL-4 released by purified neutrophils or by contaminating PBMC appeared to be very difficult because T cells represent the major source of IL-4 [13 ]. Our results showed that unstimulated PBMC released, on a per cell basis, about 60 times more IL-4 than cultured neutrophils. However, after stimulation with calcium ionophore, which is a good inducer of cytokine secretion for granulocytes but not for lymphocytes, a significant increase in the release of IL-4 was only detected in the neutrophil population. Taken together these results suggest that neutrophils are able to release small amounts of IL-4 upon activation by calcium ionophore.
In vitro stimulation with LPS contributes to the secretion
of different cytokines during the first 24 h of culture. However,
few studies have addressed the question of time-dependent production
and secretion of different cytokines or chemokines by the same PMN
[3
, 4
, 22
]. Even if the
kinetics of mRNA synthesis are quite different from one cytokine to the
other, they all lead to an increase in secretion within the first
24 h [3
]. More surprising is the fact that, under
these in vitro culture conditions, PMN could release both
pro-inflammatory cytokines like TNF- [23
] and IL-1ß
[22
] and anti-inflammatory mediators like TNF receptors
[9
] and IL-1ra [11
]. About three times
more IL-1ra was released by LPS-stimulated PMN as IL-1ß
[22
]. Furthermore, under LPS or TNF- stimulation,
IL-4 favors IL-1ra secretion while inhibiting IL-1ß production by PMN
[11
, 22
]. The production of
anti-inflammatory cytokines like IL-1ra in an inflammatory context
(LPS, TNF-), does favor the notion of a controlled release of
inflammatory cytokines by PMN. It has been reported that neutrophils do
release large amounts of IL-8 upon stimulation with LPS
[24
]. Because IL-8 has been shown to reduce the IL-4
synthesis by T cells [25
], it might be possible that
endogenous IL-8 could also regulate the production of IL-4 by
neutrophils. This could explain the dichotomy observed between the low
amounts released in the supernatant and the impressive increase in
intracellular IL-4 observed in the presence of Brefeldin A. This
compound not only blocks the release of IL-4 but also other cytokines
like IL-8, which may down-regulate IL-4 under normal in
vitro conditions.
Regarding the presence of mRNA encoding IL-4 in neutrophils, reverse
transcriptase-polymerase chain reaction (RT-PCR) and RNA protection
assays have been performed. Whereas two rounds of PCR were needed to
detect the amplified cDNA (data not shown), one cannot exclude that
even a very low proportion of contaminating T cells could be
responsible for the observed signal. Indeed, after a single round of
PCR mRNA coding for the T cell receptor, CD3 could already be detected
(data not shown). Because we needed 100 to 200 x 106
neutrophils to show IL-4 mRNA expression by RNA protection assay, a
contamination by 0.5% of T cells would represent 0.5 to 1 x
106 cells: enough to produce a signal for IL-4 (data not
shown). Furthermore, on a per cell basis, T cells contain more total
RNA as neutrophils, terminally differentiated cells, the main function
of which is not to produce cytokines but rather to take part in host
defense through oxidative burst or phagocytosis. In fact, even for
chemokines like IL-8 or GRO, PMN do not appear to be the major
source when compared to PBMC [26
]. For IL-12 subunits
p35 and p40 it has been possible to distinguish between synthesis by
neutrophils or by contaminating monocytes due to diverging responses of
these two populations to their stimulation [7
].
Unfortunately, calcium ionophore is a poor inducer of mRNA synthesis
and therefore not useful to distinguish between IL-4 mRNA production by
PMN or contaminating PBMC.
In conclusion, the present results suggest that low amounts of IL-4 can be released by neutrophils upon activation. They also lead to the concept that, because neutrophils express the IL-4 receptor [27 ], IL-4 secreted by neutrophils, even in low amounts, could exert autocrine functions. Several studies have described the effect of IL-4 on neutrophil differentiation, activation, and survival [28 29 30 ]. In neutrophils, IL-4 can induce cytoskeletal rearrangements and de novo protein synthesis [30 ]. Furthermore, in an inflammatory environment, IL-4 does enhance neutrophil respiratory burst and phagocytic activities [30 ]. Even if IL-4 did not seem to reduce cell death at 48 h as assessed by trypan blue, during the first 18 h of culture IL-4 significantly delayed neutrophil apoptosis [30 , 31 ]. It is also interesting to speculate that autocrine IL-4 could partly inhibit IL-8 production by newly recruited neutrophils [32 ] and further stimulate IL-1ra release [11 ], leading to a down-regulation of the inflammatory process. However, most of these studies have been conducted in vitro with large amounts of IL-4. The in vivo picture, where multiple cytokines and adhesion molecules can interact, is likely to be somewhat different. Therefore future studies will have to assess the importance of autocrine and paracrine secretion of low levels of cytokines. Promising approaches could involve cytokine receptor activation and interaction studies as well as dissecting transduction pathways.
Received March 31, 1999; revised February 24, 2000; accepted February 25, 2000.
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