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*
Clinical Services Program, SAIC Frederick, and
Data Management Services, National Cancer Institute, Frederick, MD 21702; and
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and -1
and monocyte
chemoattractant protein-1. In contrast, in the presence of
fibrinogen, the steady-state level of neutrophil IL-8 mRNA increased
8-fold with 5 x 10-9 M fMLP but was not decreased
with 1 x 10-7 M fMLP, suggesting that neutrophils
are specifically adapted to modulate neutrophil IL-8 synthesis through
transcriptional and posttranscriptional mechanisms. The data indicate
that fibrinogen can function not only as a substrate in the clotting
cascade, but also as an important effector during the evolution of the
innate immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(CXC) subfamily of chemokines and a
potent chemoattractant (ED50 = 1–10 ng/ml) for
neutrophils in vitro (1) as well as in vivo
(2). In an experimental human model of inflammation in
vivo, chemotactic levels of IL-8 accumulate locally (3).
The exact source of IL-8 in this model is unknown but may include
macrophages, lymphocytes, fibroblasts, and keratinocytes, which can be
induced to make IL-8, as well as exudative neutrophils, which, compared
with peripheral blood cells, have greatly increased cellular IL-8 that
is spontaneously released in vitro (4). Local release of
IL-8 by exudative neutrophils could amplify the inflammatory response
by autocrine and/or paracrine signaling. However, regulation of IL-8
production in exudative neutrophils is poorly understood. It has been
shown that treatment of peripheral blood neutrophils in vitro with an
inhibitor of the endoplasmic reticulum
Ca2+-ATPase, thapsigargin, results in a
Ca2+-dependent induction of IL-8 mRNA and
subsequent production of IL-8 to levels that are comparable to those
found in exudative neutrophils (5).
fMLP and leukotriene B4
(LTB4)3
have long been recognized as potent chemoattractants for neutrophils in
vitro and may play an important role during exudation in vivo. Low
doses of fMLP (with an optimum concentration of 
1 x
10-8 M) cause chemotaxis of neutrophils through
a specific receptor, whereas higher doses (1 x
10-7–1 x 10-6 M)
are associated with activation of the NADPH oxidase and release of
granule enzymes (6). LTB4 is also
chemotactically active through a specific receptor (7) at
a concentration of 1–100 nM, although it is less capable of activating
NADPH oxidase and degranulation than formylated peptides
(8).
Fibrinogen (Fib), a 340-kDa protein made in the liver and found
in plasma at a concentration of 1.90–3.65 mg/ml (9), is a
complex molecule composed of three pairs of polypeptide chains, an
-chain (67 kDa), a -chain (56 kDa), and a 
-chain (47 kDa). The
proteolytic action of thrombin on Fib promotes the polymerization of
the fibrin monomers, leading to fibrin clot formation. During the acute
phase response, the plasma concentration of Fib is elevated 50% within
24 h and remains elevated 3-fold over a 3-wk period
(10). Although the role of Fib in the clotting cascade has
been well defined, its role during the acute phase response is less
understood. It has been reported that mice subjected to systemic Fib
depletion and then challenged with Bacteroides fragilis
develop abdominal abscesses that are reduced in size and less purulent
than those in control mice (11), demonstrating that Fib
plays an important role in cell exudation. In this report, we show that
Fib acts synergistically with fMLP and LTB4 to
induce neutrophil IL-8 production. The data suggest Fib has an
important effector role in innate immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from the following sources: C-reactive
protein, C5a, ceruloplasmin, collagen, fibronectin, fMLP, human IgG,
laminin, LTB4, thrombin, transferrin,
vitronectin, and
N-tert-butyloxycarbonyl-L-methionyl-L-leucyl-L-phenylalanine
(N-t-BOC-MLF) were obtained from Sigma-Aldrich
(St, Louis, MO); growth-related oncongene (GRO)- was obtained from
R&D Systems (Minneapolis, MN); Fib, fibrinopeptides A and B, and Fib D
and E were obtained from Calbiochem (La Jolla, CA); serum amyloid A was
obtained from BioSource International (Camarillo, CA); cyclosporin A
and LTB4 dimethylamide were obtained from Biomol
(Plymouth Meeting, PA); indo-1 AM and calcein AM were obtained from
Molecular Probes (Eugene, OR); pertussis toxin was obtained from List
Biological Laboratories (Campbell, CA); and Sephacryl S-300 was
obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Human
recombinant Fib synthesized in Chinese hamster ovary cells
(12) was obtained from Dr. S. T. Lord (University of
North Carolina, Chapel Hill, NC). All other chemicals used were
of reagent grade and were purchased from Sigma-Aldrich. Stock
thapsigargin was dissolved in tissue culture grade endotoxin-free DMSO
at 1 mM and was stored until use at -80°C. Fib was dissolved in
sterile water, and the isotonicity was restored with 10x PBS to a
working concentration of 20–25 mg/ml. Analysis of commercial Fib by
SDS-PAGE (Invitrogen, Carlsbad, CA) under reducing conditions and
subsequent silver staining resulted in primarily the (67 kDa),
(56 kDa), and (47 kDa) subunits of Fib, verifying the purity of the
Fib preparation. Fib levels were determined by radial immunodiffusion
assay (The Binding Site, San Diego, CA). Commercial Fib contained a
trace of contaminating endotoxin (1 pg endotoxin/mg of Fib as
determined by a chromogenic Limulus amebocyte assay
(BioWhittaker, Walkersville, MD); however, treatment of
neutrophils with LPS (up to 100 ng/ml, E. coli serotype
0111:B4; Sigma-Aldrich) did not duplicate the synergy observed with Fib
and fMLP or LTB4, indicating that the results
described in this report could not be attributed to the contaminating
endotoxin. In addition, commercial Fib contained trace contamination
with human serum albumin (<40 ng albumin/mg of Fib as determined by a
human albumin ELISA (Bethyl Laboratories, Montgomery, TX); this
concentration of albumin had no effect on the neutrophil functions
studied. Analysis of recombinant human Fib by SDS-PAGE under reducing
conditions and subsequent silver staining resulted in three predominant
protein bands that comigrate with the , , and subunits of
commercial Fib.
Isolation of peripheral blood neutrophils
Peripheral blood (anticoagulant citrate dextrose solution USP formula A; Baxter Healthcare, Deerfield, IL) was obtained from normal volunteers through the Frederick Research Donor Program (Protocol OH99-C-N046). Neutrophils were isolated by centrifugation through a discontinuous Ficoll-Hypaque density gradient and sedimentation with dextran as described previously (4). The final preparation of neutrophils was 95% pure, with 4% eosinophils and >95% pure, with 4% eosinophils and <1% monocytes and lymphocytes, as assessed by differential staining.
IL-8 production by peripheral blood neutrophils
Neutrophils (2 x 106) were suspended in HBSS with 10 mM HEPES (pH 7.3) and incubated in polypropylene tubes for up to 8 h at 37°C in the presence or absence of either chemoattractant or Fib. After incubation, the cell suspensions were spun at 4°C and the supernatant fluid harvested for analysis. Cell pellets were solubilized in 0.2% Triton X-100 using 20 1-s pulses (minimum setting) with a microtip sonicator (Sonifier II; Branson Ultrasonics, Danbury, CT).
Assays
All of the cytokines were measured using either commercial
enzyme-linked immunosorbent assays (IL-1, IL-1 receptor antagonist
(IL-1ra), IL-8, macrophage inflammatory protein (MIP)-1, MIP-1,
and TNF-; R&D Systems) or laboratory enzyme-linked immunosorbent
assays using matched Abs (R&D Systems) that were prepared according to
the manufacturer’s recommendations. The manufacturer’s limits of
detection are as follows: IL-1, 1 pg/ml; IL-1ra, 14 pg/ml; IL-8, 10
pg/ml; MIP-1, 6 pg/ml; MIP-1, 4 pg/ml; and TNF-, 4 pg/ml.
Intracellular staining for IL-8
Neutrophils (2 x 106/ml HBSS-HEPES) were incubated in the presence or absence of Fib (2 mg/ml) and fMLP (5 x 10-9 M) for 4 h at 37°C. Because the majority of the neutrophil-derived IL-8 remained cell-associated, no blockers of Golgi transport were used to enhance the response. The cells were then fixed, stained using a commercial intracellular staining kit (BioSource International), and analyzed by flow cytometric analysis.
Gel filtration and PAGE analysis of Fib
Fib (20 mg) was loaded on a high resolution Sephacryl S-300 column (1.6 x 60 cm; Amersham Pharmacia Biotech) and eluted with PBS at a flow rate of 0.5 ml/min with 1.5-ml fractions. The column was calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), BSA (67 kDa), OVA (43 kDa), and RNase (14 kDa). Fractions were analyzed on precast NuPAGE 4–12% gels, and stained with a SilverXpress silver staining kit (Invitrogen).
Treatment with pertussis toxin
Neutrophils (2 x 107/ml of HBSS-HEPES) were treated in the absence or presence of pertussis toxin (1µg/ml) for 3 h at 37°C, washed twice with HBSS without divalent cations, and resuspended in HBSS-HEPES before use. Pertussis toxin treatment reduced fMLP-induced O2 production by 85% without altering the PMA-induced response.
Determination of changes in intracellular Ca2+ concentration ([Ca2+]i)
Neutrophils (1 x 107/ml HBSS-HEPES)
were incubated with the cell-permeant dye, indo-1 AM, in the dark at
37°C for 45 min. The neutrophils were then pelleted by centrifugation
at 200 x g, resuspended in HBSS-HEPES, and the
procedure was repeated to remove remaining extracellular indo-1 AM. The
cells were resuspended at 2.5 x 106/ml
HBSS-HEPES. Changes in
[Ca2+]i were monitored on
a DeltaScan spectrophotometer (Photon Technology International,
Lawrenceville, NJ) using a thermostatically controlled cuvette holder.
Data were collected as the ratio (R) of the 
emissions
(402 nm/486 nm) where
excitation was at 358 nm.
[Ca2+]i was calculated as
previously described (13). The parameters
Rmax and Rmin
were determined empirically by addition of ionomycin (1 µM) and EGTA
(12.5 mM), respectively.
O2- production by cytochrome c reduction
Neutrophils (1 x 106/ml HBSS) were preincubated at 37°C for 15 min in the absence or presence of Fib (2 mg/ml). Cytochrome c (150 nmol) was added to each tube before the addition of the indicated concentration of fMLP. The incubation was continued for 30 min. Superoxide dismutase (100 µg/ml) was added to an identical tube as a blank. Samples were analyzed using an analytical wavelength at 549.5 nm and background wavelengths at the isobestic points, 541.0 and 556.0 nm. A micromolar extinction coefficient of 0.0211 was used to convert absorbance values to nanomoles of O2- released.
Release of granular enzymes
Neutrophils (2 x 106/ml of HBSS with 10 mM HEPES) were incubated in the presence and absence of Fib for 15 min before the addition of fMLP. After 8 h, the cells were isolated from the cell suspension by centrifugation at 200 x g at 4°C. Both the neutrophil pellet and the supernatant fraction were analyzed for granular enzyme content. Data are expressed as the percentage of the total found in the supernatant fraction.
Lactoferrin was determined by an ELISA as previously described
(14). Levels of -D-glucuronidase
were determined by measuring the release of 4-methylumbelliferone
from 4-methylumbelliferyl--D-glucuronide. Briefly, an
aliquot of the sample (0.01 ml) was incubated with 5 mM substrate in
100 mM sodium acetate buffer (pH 4.8) for 30 min at 37°C (final
volume of 0.05 ml). The reaction was terminated by the addition of 0.2
ml of ammonium glycine buffer (0.2 M NH4OH and
0.05 M glycine (pH 10.5)), and the fluorescence of the samples was
determined using a CytoFluor II 96-well fluorescence plate reader
(PerSeptive Biosystems, Framingham, MA).
-D-Glucuronidase activity was determined using a
4-methylumbelliferone standard curve.
Chemotaxis
Chemotaxis was determined using calcein AM-loaded neutrophils and a disposable 96-well chemotaxis chamber (NeuroProbe, Gaithersburg, MD) as previously described (15). A polycarbonate filter with a pore size of 8 µm and a pore density of 1000 pores/mm2 was used for the analysis. Migrating neutrophils were suspended in the presence or absence of Fib (2 mg/ml) before migration through the filter. After incubating the filter chamber at 37°C for 60 min, the top of the filter was washed three times with HBSS without divalent cations. The fluorescence of the filter and plate was determined on a CytoFluor II fluorescence plate reader. The number of migrating cells was determined using a standard curve of calcein-labeled neutrophils. In control studies, there was <10% spontaneous release of calcein.
Analysis of neutrophil mRNA
Neutrophils (2.5 x 106/ml HBSS with
10 mM HEPES) were incubated for 1 h and then harvested by
centrifugation at 200 x g at room temperature. Total
cellular RNA was isolated using TRIzol (Life Technologies,
Gaithersburg, MD). The OD260 nm/OD280
nm of the extracted RNA was usually 1.6; total RNA yields
were 20–30 µg/5 x 107 neutrophils. RNA
concentrations were determined using the RiboGreen RNA quantitation
system (Molecular Probes). The RiboQuant RNase protection assay kit and
the hCK-5 RiboQuant human cytokine multi-probe template set (BD
PharMingen, San Diego, CA) were used according to the
manufacturer’s instructions with the exception that the probes labeled
with [32P]UTP (Amersham, Arlington Heights, IL)
were purified using TE Micro Select-D, G-25 microcentrifuge spin
columns (5 Prime
3 Prime, Boulder, CO). Protected fragments were
resolved on 6% polyacrylamide sequencing gels and quantitated on a BAS
1000 phosphor imager (Fugi Medical Systems, Fairfield, NJ). However, to
measure steady-state mRNA levels for IL-8, MIP-1, and MIP-1, the
amount of total RNA added was reduced to 5 ng (the manufacturer’s
recommendation is 1–20 µg) to avoid saturation of these components
of the multi-probe template set. Higher concentrations of total
RNA that were run initially demonstrated the integrity of the RNA
from all samples and yielded approximately equal signals for the GAPDH
housekeeping gene and L32 ribosomal RNA probe. The RNA concentrations
of the diluted samples were confirmed by the RiboGreen RNA
quantification system.
Statistics
Data were evaluated with a variety of statistical techniques including linear and nonlinear regression, ANOVA, analysis of covariance, repeated-measures analysis, profile analysis, descriptive techniques, and post hoc tests. In many cases, data were transformed to their common logarithms to satisfy homogeneity of variance requirements. The data presented represent the mean ± SEM.
A conservative strategy was used to define synergy. Following global tests of significance, if the treatment of neutrophils with both Fib and chemoattractant yielded a significantly higher mean response (i.e., p < 0.01) than Fib alone and a significantly higher mean response than chemoattractant alone, then the effect of Fib and chemoattractant were defined to be synergistic. As a further validation, the aggregate response of Fib alone and of chemoattractant alone was compared with the response of both ligands, and similar significance was observed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly isolated neutrophils have low, but detectable,
levels of IL-8 (0.04 ± 0.01 ng/106 cells)
(Fig. 1
A; t = 0). As
shown previously (4), incubation of neutrophils in buffer
alone for 8 h resulted in a small but significant increase in the
level of total IL-8 (sum of the cellular and extracellular) compared
with freshly isolated neutrophils. Treatment of neutrophils for 8
h with a chemotactic concentration of the peptide fMLP (5 x
10-9 M) alone (Fig. 1A), or a
chemotactic concentration of LTB4 (1 x
10-7 M) alone (Fig. 1B), resulted in
small increases in the levels of total IL-8 compared with freshly
isolated neutrophils (buffer treated = 0.36 ± 0.04 ng
IL-8/106 cells, p < 0.001; fMLP
treated = 1.14 ± 0.13 ng IL-8/106
cells, p < 0.01; and LTB4
treated = 3.42 ± 0.77 ng IL-8/106
cells, p < 0.001). Thus, fMLP and
LTB4 are able to induce neutrophil IL-8
production, but not at levels that could account for the observed
production of IL-8 in exudative neutrophils.
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A). Similarly, treatment of neutrophils with both Fib and
LTB4 (1 x 10-7 M)
resulted in an increase in total IL-8 that was detected within 1 h
and continued to increase through 8 h. Fib plus
LTB4 yielded significant synergistic responses vs
neutrophils treated with Fib alone or LTB4 alone
at t = 1, 2, 4, and 8 h (p
< 0.01 at all time points) (Fig. 1B). Dose response of Fib
Exposure of neutrophils to increasing concentrations of Fib alone
(1–5 mg/ml) resulted in small (p < 0.05)
increases in total IL-8 (Fig. 1C). Treatment of neutrophils
with increasing concentrations of Fib plus a constant concentration of
fMLP (5 x 10-9 M) greatly increased
neutrophil IL-8 at all doses of Fib tested (p
< 0.01). Similarly, treatment of neutrophils with increasing
concentrations of Fib plus LTB4 (1 x
10-7 M) increased neutrophil IL-8 at 0.5–5
mg/ml of Fib (p < 0.01) (Fig. 1D).
Removal of Fib by washing the cells abrogated its synergistic effect on
IL-8 production, yielding a response comparable to that of fMLP alone
(data not shown).
Dose response of fMLP and LTB4
Incubation of neutrophils with Fib and increasing concentrations
of fMLP yielded an approximately bell-shaped curve with maximum IL-8
production at 2–5 x 10-9 M fMLP
(ED50 = 1.15 ± 0.05 nM), doses associated
with chemotaxis (16) and binding to the high affinity fMLP
receptor (Fig. 1E). A striking reduction in neutrophil IL-8
was observed with higher doses of fMLP (up to 1 x
10-7 M), indicating sensitive regulation of
neutrophil IL-8 synthesis. A second peak of IL-8-inducing activity,
exhibiting marginal synergy (p < 0.05), was
observed in the range of 5 x 10-6–1
x 10-5 M fMLP, doses associated with binding of
fMLP to the low affinity fMLP receptor (17). The
dose-response characteristics of IL-8 synthesis in response to Fib plus
fMLP are similar to the neutrophil chemotactic response to
fMLP.
The dramatic decrease in IL-8 production observed at doses of 5 x 10-9–1 x 10-7 M fMLP was not due to increased proteolytic activity in the extracellular fluid. In experiments in which exogenous 125I-labeled IL-8 was added to neutrophils exposed to Fib plus fMLP (5 x 10-9 M) for 8 h and then the cell-free supernatant fluid analyzed by SDS-PAGE and autoradiography, there was no degradation of IL-8 (data not shown). In addition, treatment of neutrophils with a higher dose of fMLP (1 x 10-7 M) 4 h after treatment with optimal conditions of Fib plus fMLP (5 x 10-9 M) resulted in no decrease in IL-8 production, indicating that no mediators were released at a higher dose of fMLP that degraded newly synthesized IL-8.
Treatment of neutrophils with Fib plus LTB4 also
resulted in significant synergistic responses at doses of 2 x
10-8–1 x 10-7 M
(Fig. 1F; p < 0.01 at all doses). However,
in contrast to the response with fMLP, higher doses of
LTB4 (up to 10-6 M) were
not associated with reduced levels of IL-8. No increase in neutrophil
IL-8 was observed when neutrophils, in the presence or absence of Fib,
were incubated with the chemoattractants platelet-activating factor
(PAF), complement fragment, C5a, or GRO- (Table I). Because GRO- and IL-8 share a
common CXCR (CXCR-2), the failure of GRO- to synergize with Fib
indicates that IL-8 does not affect its own synthesis through
CXCR-2.
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To determine whether the increases in neutrophil IL-8 induced by
Fib plus fMLP and Fib plus LTB4 were mediated by
their respective chemoattractant receptors, neutrophils were treated
with either N-t-BOC-MLF, a competitive antagonist
of fMLP (18), or LTB4-dimethylamide,
a competitive antagonist of LTB4
(19). Treatment with these receptor antagonists abrogated
the fMLP- and LTB4-induced responses by 75 and
70%, respectively (Fig. 2), but had only
minor effects on the thapsigargin-induced increase in neutrophil
IL-8.
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To determine whether the increases in neutrophil IL-8 induced
through the fMLP receptor and LTB4 receptor were
mediated byheterotrimeric G proteins, neutrophils were
pretreated with pertussis toxin to inactivate the G proteins before the
addition of agonists. As shown in Fig. 3, treatment of neutrophils with pertussis toxin reduced the responses to
Fib plus fMLP and Fib plus LTB4 to the level
observed with Fib alone. However, the increases in neutrophil IL-8 in
response to buffer vs Fib alone (p < 0.01) or
to thapsigargin alone (p < 0.001) were not
significantly altered by treatment with pertussis toxin, indicating
that signaling through these agonists was independent of G
proteins.
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To determine the specificity of the response to Fib, other
extracellular matrix proteins including human fibronectin, vitronectin,
collagen, and laminin were tested and failed to mimic the effect of Fib
on IL-8 production (Fig. 4). Of the
acute-phase proteins tested, ceruloplasmin and C-reactive protein both
mimicked the effect of Fib, but to a lesser extent.
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Heat-treated Fib (30 min at 56°C) and a fibrin clot were unable
to synergize with fMLP to induce IL-8 production (Table II). The fibrinopeptides A and B,
released from Fib by thrombin during the clotting process, and the
proteolytic Fib fragments D (85 kDa) and E (50 kDa), derived from the
complete hydrolysis of Fib, were also unable to support the production
of IL-8 from neutrophils stimulated with fMLP.
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A). PAGE analysis of the peak
fractions under reducing conditions yielded three bands, consistent
with the three subunits of Fib (Fig. 5, shown as insert). The addition
of thrombin to Fib to promote clot formation and the subsequent removal
of the fibrin clot by centrifugation resulted in loss of the three Fib
subunits and abrogation of the IL-8 response, indicating that Fib was
responsible for synergistic induction of neutrophil IL-8 production
(Fig. 5B). To exclude the possible contribution of a
contaminant(s) in the native Fib, recombinant Fib was also tested. In a
single experiment, treatment of neutrophils with human recombinant Fib
(2 mg/ml) and fMLP resulted in a 4-fold increase in neutrophil IL-8
compared with neutrophils treated with recombinant Fib alone. Although
the response to recombinant Fib was somewhat reduced compared with
native Fib (7-fold increase), its effectiveness excluded a contaminant
of the commercial Fib as the active agent.
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To address whether a specific subpopulation of neutrophils or some other contaminating cell type contributed to increased levels of IL-8, neutrophils were permeabilized and stained for intracellular IL-8. Neutrophils treated with Fib (2 mg/ml) and fMLP (5 x 10-9 M) responded as a single population of cells with an apparent shift in the peak of intracellular IL-8 staining to the right as compared with untreated (control) cells (data not shown).
Effect of Fib on other neutrophil functions
The synergy observed with Fib plus fMLP was not observed for other
neutrophil functions. Fib had no effect on the dose-response curve of
fMLP for alterations in
[Ca2+]i, chemotaxis,
O2 production, or degranulation of either specific
(lactoferrin) or azurophilic
(-D-glucuronidase) granules (data not
shown).
Studies of neutrophils from patients with leukocyte adhesion deficiency (LAD)
Fib responses in neutrophils have been attributed to interactions
with the 2 integrins (20, 21). To
investigate the potential role of the 2
integrins in mediating the effects of Fib on neutrophil IL-8
production, neutrophils were isolated from patients who lack the
2 integrins (LAD). LAD neutrophils supported
Fib plus fMLP-induced IL-8 production normally, indicating that the
2 integrins were not the receptors mediating
the Fib effects (Table III).
Interestingly, treatment of LAD neutrophils with fMLP in the absence of
Fib resulted in an IL-8 response that was significantly elevated vs
normal subjects.
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The levels of IL-8 attained after treatment of neutrophils with
Fib plus fMLP (12.48 ± 1.11 ng IL-8/106
cells) and Fib plus LTB4 (23.01 ± 2.31 ng
IL-8/106 cells) were comparable to concentrations
in exudative neutrophils (4) and thapsigargin-treated
neutrophils (5). The Ca2+ chelator,
EGTA (2.5 mM), inhibited the Fib plus fMLP response by <50%, whereas
the calcineurin inhibitor, cyclosporin A (200 ng/ml), had no effect,
indicating that the neutrophil response to Fib plus fMLP, unlike the
response to thapsigargin (5), was not dependent on
Ca2+ (Table IV).
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The production of IL-8 by Fib plus fMLP was dependent on protein synthesis de novo. Treatment of neutrophils with cycloheximide (10 µg/ml) inhibited the production of IL-8 with Fib plus fMLP by 89% (8.69 ± 2.15 ng/1 x 106 cells vs 0.95 ± 0.33 ng/1 x 106 cells; p = 0.0004). IL-8 production induced by Fib plus fMLP was inhibited (89%) by the RNA synthesis inhibitor, actinomycin D (10 µg/ml) (22.23 ± 8.19 ng/1 x 106 cells vs 2.61 ± 0.15 ng/1 x 106; p = 0.0368), a dose which has been shown to block completely the increased expression of IL-8 mRNA (22) and formyl peptide receptor mRNA (23) in neutrophils.
The induction of IL-8 synthesis was accompanied by changes in the
steady-state level of IL-8 mRNA. Using RNase protection assays of total
RNA isolated from neutrophils, the level of IL-8 mRNA could be detected
using as little as 5 ng of total neutrophil RNA, much less than usually
needed for RNase protection assays. Analysis of neutrophil RNA revealed
that treatment of neutrophils with chemotactically active
(16) doses of fMLP (5 x 10-9
M) alone, LTB4 (1 x
10-7 M) alone, or Fib alone had little effect on
the steady-state level of IL-8 mRNA compared with untreated neutrophils
incubated for 1 h (Fig. 6), although
small, but significant, increases in neutrophil IL-8 were observed
(Fig. 1, A and B). At a high dose of fMLP alone
(1 x 10-7 M), associated with reduced
chemotactic responsiveness (16), there was a 3-fold
increase in the steady-state level of IL-8 mRNA but no further increase
in neutrophil IL-8 synthesis. Addition of Fib to fMLP (5 x
10-9 M) or LTB4 (1 x
10-7 M) resulted in an 8- or 4-fold increase in
the steady-state level of IL-8 mRNA, respectively, and a 30-fold
increase in neutrophil IL-8 (Fig. 6). The complete inhibition of IL-8
synthesis observed with Fib plus fMLP at 1 x
10-7 M vs Fib plus fMLP at 5 x
10-9 M (Fig. 1E) could not be
explained by a reversal in the steady-state level IL-8 mRNA (Fig. 6),
indicating that additional regulatory mechanisms were involved in
down-regulating IL-8 synthesis with 1 x
10-7 M fMLP.
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). Treatment of neutrophils with
Fib plus LTB4 (1 x
10-7 M) yielded a lower steady-state level of
IL-8 mRNA than Fib plus fMLP (5 x 10-9 M),
but the net production of IL-8 was higher with
LTB4 than fMLP, indicating additional
ligand-specific differences in posttranscriptional regulation of IL-8
synthesis.
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and MIP-1 was not detected in freshly isolated cells, yet it was
detected in low amounts upon incubation for 1 h without a specific
stimulus (Fig. 7). No further increases in the steady-state levels of
MIP-1 and MIP-1 mRNA were observed under the various treatment
regimens. The failure to induce changes in the steady-state levels of
MIP-1 and MIP-1 mRNA and the minimal changes in the synthesis of
other neutrophil cytokines, IL-1, TNF-, and IL-1ra (Table V), attest to the specificity of the IL-8
response to Fib plus fMLP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The synergistic induction of neutrophil IL-8 synthesis is specific for
Fib plus fMLP or LTB4; no synergy is observed
with Fib plus the chemoattractants C5a, PAF, and GRO-. Because
GRO- and IL-8 share a common CXCR (CXCR-2), the failure of GRO-
to synergize with Fib suggests that IL-8 does not affect its own
synthesis through CXCR-2. However, it does not preclude IL-8 mediation
of its own synthesis through CXCR-1. Browning et al. (24)
recently reported that immobilized IL-8 induced IL-8 production in
monocytes; however, these authors failed to demonstrate any significant
IL-8 production in neutrophils. Furthermore, these authors showed that
the stimulation of neutrophils through CXCR-2 (with immobilized
GRO-) did not induce IL-8 production, confirming the results
presented in Table I. The synergistic increases in neutrophil IL-8
observed with both Fib plus fMLP and Fib plus
LTB4 are sensitive to specific receptor
antagonists and pertussis toxin and are therefore mediated by their
respective G protein-coupled chemoattractant receptors. In contrast,
the modest increase in neutrophil IL-8 observed with Fib is insensitive
to pertussis toxin and is therefore independent of a G protein-coupled
receptor-signaling event.
The synergy of Fib plus fMLP is limited to IL-8 synthesis because other fMLP-mediated neutrophil functions (chemotaxis,O2 production, and degranulation) are not altered in the presence of Fib.
The effect of Fib on IL-8 production is both dose dependent and specific. The effective doses of Fib range from the levels of Fib found in normal plasma to the levels found during the acute-phase response. Heat-treated Fib, fibrinopeptides A and B (proteolytic fragments of Fib released during clot formation), as well as a fibrin clot fail to exhibit synergy with fMLP for enhanced neutrophil IL-8 production. Analysis of Fib by gel filtration chromatography results in a single peak of IL-8-inducing activity that coelutes with both the peak of protein activity and the peak of Fib activity. Removal of the Fib by treatment with thrombin abrogates the IL-8-inducing activity, supporting the role of Fib in inducing neutrophil IL-8 production. Finally, the effectiveness of recombinant Fib in inducing neutrophil IL-8 production excludes a contaminant of Fib in our commercial preparation as the active agent and further supports the role of Fib. The reduced response of recombinant Fib compared with native Fib probably reflects glycosylation differences of the recombinant protein.
Extracellular matrix proteins such as vitronectin, fibronectin, laminin, collagen, and thrombospondin and acute-phase proteins such as serum amyloid A and transferrin are unable to mimic the effects of Fib. The acute-phase proteins, ceruloplasmin and C-reactive protein, are able to mimic the synergy observed with Fib, although the extent of IL-8 production is diminished. Interestingly, elevated levels of Fib and C-reactive protein are predictors of long-term risk of death from cardiac causes (25), and it is intriguing to speculate that the observation presented in this report may relate to the pathogenesis of atherosclerosis.
Using RNase protection assays of total RNA isolated from resting
neutrophils, the mRNA for IL-8, MIP-1, and MIP-1 was detected.
Stimulation of neutrophils with Fib plus fMLP or Fib plus
LTB4 increased the steady-state levels of IL-8
mRNA, but not MIP-1 and MIP-1. The increase in protein
translation was relatively specific, with a 30-fold increase in the
levels of IL-8 compared with control, a 4-to 6-fold increase in the
level of IL-1 and TNF-, and little or no increase in the levels
of IL-1ra, MIP-1, and MIP-1. The preferential induction of
neutrophil IL-8 vs other cytokines/chemokines indicates that
neutrophils are specifically adapted to amplify IL-8 synthesis and
suggests that there are complex regulatory mechanisms involved.
Fib plus fMLP induction of neutrophil IL-8 was tightly regulated, with a biphasic dose-response curve. Low doses of fMLP (5 x 10-9 M) caused a 30-fold increase in IL-8 synthesis, whereas at a higher dose (1 x 10-7 M), the response was ablated; increasing fMLP to 1 x 10-5 M resulted in a second smaller response. The biphasic dose-response curve is the same as the chemotactic response and may relate to high and low affinity fMLP receptors (16). The depressed IL-8 synthesis at 1 x 10-7 M compared with 5 x 10-9 M could not be attributed to the release of proteolytic enzymes degrading fMLP or IL-8 or to inhibition of IL-8 synthesis. Further investigation of the mechanism of the posttranslational regulation of IL-8 synthesis may offer important clues as to normal regulation of inflammation.
Cellular interactions with Fib are thought to be mediated through lower
affinity binding to promiscuous surface integrins rather than through
higher affinity binding via a specific Fib receptor. Fib has been shown
to bind to endothelial cells through the integrin
V1 receptor
(26) and thereby has been implicated in angiogenesis and
wound healing. Fib has also been shown to activate platelets through
the IIb3 receptor
(27) and is thought to play an important role in
activating platelet aggregation and clot retraction. Neutrophil
interactions with surface-bound Fib via 2
integrins results in a substantial increase in the production of IL-8
and IL-1, a response that is abrogated in CD18-deficient mice
(28). However, the synergistic effect of Fib plus fMLP on
neutrophils described here is independent of the
2 integrins because neutrophils from three
patients with leukocyte adhesion deficiency, whose cells lack these
receptors, exhibit responses to Fib plus fMLP that are normal
(Table III).
The production of IL-8 in vitro by neutrophils mimics that observed in exudative neutrophils and indicates that migrating neutrophils are a potential source of IL-8 found at an inflammatory focus. Moreover, this observation provides a plausible rationale for the logarithmic amplification of the inflammatory response through neutrophil IL-8-mediated recruitment of additional neutrophils. Finally, insight into the regulatory mechanisms that precipitate neutrophil IL-8 production may provide therapeutic targets for modulation of the innate immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John I. Gallin, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Building 10, Room 11N107, Bethesda, MD 20892-1504. E-mail address: jgallin{at}cc.nih.gov
3 Abbreviations used in this paper: LTB4, leukotriene B4; Fib, fibrinogen; N-t-BOC-MLF, N-tert-butyloxycarbonyl-L-methionyl-L-leucyl-L-phenylalanine; GRO, growth-related oncogene; IL-1ra, IL-1R antagonist; MIP, macrophage inflammatory protein; [Ca2+]i, intracellular Ca2+ concentration; LAD, leukocyte adhesion deficiency; R, ratio; PAF, platelet-activating factor.
Received for publication January 2, 2001. Accepted for publication June 27, 2001.
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References |
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; Fib (2 mg/ml), 
;
A, fMLP (5 x 10-9 M), 
; Fib plus
fMLP, •; and B, LTB4 (1 x
10-7 M), 
