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*
Department of Pathology and
Graduate Program in Cellular and Molecular Biology, University of Michigan Medical Center, and
Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 h) up-regulated. In contrast, no significant induction of CCR2,
CCR3, CCR4, or CCR5 mRNA was observed. CCR1 protein was also
up-regulated by GM-CSF stimulation. GM-CSF-induced up-regulation of
CCR1 showed functional consequences because GM-CSF-treated PMN, but not
control cells, responded to the CC chemokines macrophage inflammatory
protein-1
, monocyte chemoattractant protein-3, and RANTES in assays
of chemotactic migration and intracellular calcium mobilization. These
results suggest that PMN activated by the proinflammatory cytokine
GM-CSF can change their receptor expression pattern and become
responsive to CC chemokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The physiological effects of chemokines are exerted through binding of
a family of seven transmembrane receptors, which couple to
G-protein-mediated pathways within the cell (3). Ten human
CC receptors and five human CXC receptors have been identified to date
(3, 6). The expression pattern of chemokine receptors is a
major factor in dictating the selectivity of chemokines for different
target cells. Traditionally, human PMN have been thought to express
receptors from the CXC (2) or CX3C
(7, 8) family and to preferentially migrate to chemokine
ligands from only these two families (2, 9). However,
different studies have provided conflicting evidence on whether human
PMN express CCRs. Although Xu et al. (10) have found that
resting human PMN possess binding sites for the CC chemokines
macrophage inflammatory protein (MIP)-1 and monocyte chemoattractant
protein (MCP)-3, a study by McColl et al. (11) did not
detect any binding sites for CC chemokines on these cells. Furthermore,
human PMN do not respond to the CC chemokine MIP-1 in in vitro
assays of chemotaxis (11, 12). Therefore, it is generally
assumed that CC chemokines have no functional effects on resting
human PMN.
In contrast to findings in human PMN, several murine models of
inflammation have shown that CC chemokines do play a role in PMN
chemotaxis. For example, in vivo studies of LPS- and
endotoxemia-associated lung injury in mice have demonstrated that
neutralization of MIP-1 attenuates neutrophil infiltration into
inflammatory sites (13, 14). Furthermore, PMN isolated
from inflammatory exudates in mice display chemotactic migration and
calcium flux responses to MIP-1 (15).
A growing body of literature demonstrates that cytokines regulate
chemokine receptor expression on a variety of different cell types.
Pro-inflammatory mediators such as TNF-, IL-1
, and LPS
down-regulate CCR2 on human monocytes (16), while the
anti-inflammatory cytokine IL-10 up-regulates CCR1, CCR2, and CCR5
on these cells (17). Chemokine receptor expression on T
lymphocytes is also modulated by cytokines, as studies have shown that
IL-2 increases CCR1 (18), CCR2 (18), and
CXCR3 (19), IL-10 decreases CCR5 (20), and
IL-15 induces CCR1, CCR2, CCR4, and CCR5 on these cells
(21). PMN expression of CXC chemokine receptors is also
regulated by pro-inflammatory factors such as TNF (22, 23)
and LPS (16, 24), which both down-regulate CXCR1 and
CXCR2. In contrast, G-CSF (not GM-CSF) induces the expression of CXCR1
and CXCR2 on PMN (25).
The present study examines the effects of GM-CSF, a pro-inflammatory cytokine that is a potent activator of PMN and other granulocytes (26). This cytokine is already well known for its role in neutrophil maturation and priming (26, 27), as well as its ability to slow the apoptotic rate of PMN in vitro (28). In this study we demonstrate that GM-CSF induces CCR expression in PMN, suggesting that the in vivo maturation and priming of these cells involves expression of CC-type chemokine receptors, and that CC chemokines can exert direct effects upon activated human PMN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human GM-CSF, MIP-1, MIP-1, MCP-1, MCP-3,
MCP-4, RANTES, and IL-8 were purchased from PeproTech (Rocky Hill, NJ).
Recombinant human eotaxin was purchased from R&D Systems (Minneapolis,
MN). HBSS was obtained from BioWhittaker (Walkersville, MD).
Complete medium consisted of RPMI 1640 (Life Technologies, Grand
Island, NY) supplemented with 2 mM L-glutamine, 25 mM
HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (Hazelton Research
Products, Lenexa, KS), and 10% heat-inactivated FCS (Life
Technologies).
Cell isolation
PMN were isolated from human peripheral blood as previously described (29). Briefly, peripheral venous blood from healthy volunteers was drawn into heparinized syringes, mixed 1:1 with sterile saline, and layered over Ficoll-Paque (Pharmacia LKB, Piscataway, NJ) columns. These columns were centrifuged at 1500 rpm for 30 min at room temperature, the plasma and mononuclear cell layer was discarded, and the erythrocytes were eliminated by hypotonic lysis in ammonium chloride buffer. PMN were washed once in HBSS/BSA (HBSS without calcium or magnesium, with 0.1% BSA added) and resuspended in complete medium. Cell purity, assessed by Diff-Quik (Baxter, Rome, Italy) staining and light microscopy, was routinely >95% with the major contaminant being eosinophils. Viability as assessed by trypan blue exclusion was >99% for freshly isolated cells.
RNase protection assay
Total RNA was isolated from PMN using the guanidine
isothiocyanate method as previously described (30) and
used in the standard PharMingen (San Diego, CA) RNase protection
protocol as follows. The multiprobe template set hCR5, containing DNA
templates for CCR1, CCR3, CCR4, CCR5, CCR2a+b, CCR2a, CCR2b, L32, and
GAPDH, was purchased from PharMingen. This template set was used to
synthesize [-32P]UTP (Amersham,
Buckinghamshire, U.K.)-labeled probes in the presence of a GACU pool
using a T7 RNA polymerase. Probes were hybridized overnight with 5–10
µg target RNA, followed by RNase digestion and proteinase K
treatment. Samples were chloroform-extracted, ethanol precipitated in
the presence of ammonium acetate, and loaded on an acrylamide-urea
sequencing gel made in 0.53 M Tris-borate/EDTA (TBE) buffer. After
electrophoresis at 50 watts for 1–2 h, the gel was adsorbed to filter
paper, dried under vacuum, and exposed to film (X-OMAT, Kodak,
Rochester, NY) with intensifying screen at -70°C. Alternatively, the
dried gel blot was exposed to a phosphorscreen for phosphorimagery
analysis using ImageQuant software. The intensity of each band was
adjusted for the number of UTPs present in each of the probe sequences.
These adjusted values were normalized to the adjusted values of the
housekeeping gene L32, and normalized values were used to quantify
expression as fold-increase over control.
Protein isolation and Western blot analysis
Protein extracts were isolated from cultured neutrophils as follows: 2 x 107 cells were pelleted and washed once with PBS and once in buffer A (10 mM HEPES, 10 mM KCl, 0.5 mM DTT). The cell pellet was lysed in 20 µl buffer A plus 1% Nonidet P-40 and incubated for 10 min at 4°C. Samples were then spun at 16,000 x g in a microcentrifuge for 2 min. The supernatant was removed, mixed with 55 µl buffer C (20 mM HEPES, 500 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 1.5 mM MgCl2, 20% glycerol), and saved as cytoplasmic extract. One-hundred micrograms of protein per sample was analyzed by Western blot. Proteins were separated by 12% SDS-PAGE and blotted onto Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) by electrotransfer. Membranes were blocked with 5% milk and stained with rabbit polyclonal Abs raised against human CCR1 or CCR3 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by a peroxidase-conjugated goat Ab against mouse IgG (Pierce, Rockford, IL). Signals were visualized by chemiluminescence using super signal reagents (Pierce).
Measurement of intracellular free calcium
Intracellular calcium levels were monitored using standard fluorometric techniques (31). Cells were loaded with the fluorescent dye fura-2AM (Sigma, St. Louis, MO) as follows: PMN were suspended in HBSS/BSA (without Ca+2 or Mg+2) containing 2.5 µM fura-2AM and incubated at 37°C for 30 min. Cells were washed three times in HBSS/BSA and resuspended in same buffer with addition of 1 mM Mg+2 and 0.1 mM Ca+2. Extracellular Ca+2 was restored to 1 mM immediately before the assay. Fluorescence was monitored at the excitation wavelengths 340 and 380 nm and emission 510 nm in a Perkin-Elmer (Norwalk, CT) LS 50B luminescence spectrometer using FL WinLab software (Perkin-Elmer). Baseline fluorescence was monitored for 30 s before the addition of agonists in small volume (<50 µl). Calcium index is expressed as the ratio of emission intensity at excitation wavelength 340 nm divided by emission excited at 380 nm.
Chemotaxis assays
Chemotactic activity was monitored using a chemotaxis microchamber technique as previously described (32). Briefly, chemoattractant solution or control medium (HBSS containing Mg+2 and Ca+2 plus 0.1% BSA) was added to the lower wells of a 12-well chemotaxis chamber (Neuroprobe, Pleasanton, CA), a polycarbonate polyvinylpyrrolidone-free PVPF filter with a 5-µM pore size (Poretics, Livermore, CA) layered on top, and the top plate was affixed. A cell suspension of 2 x 106 PMN/ml was seeded into the upper well and the whole chamber was incubated at 37°C in air with 5% CO2 for 60 min. Filters were removed and the upper sides were scraped clean and stained with Diff-Quik (Baxter). All treatments were performed in triplicate wells. The number of PMNs in three high-power (1000x) oil-immersion fields were counted for each well. Chemotactic index (CI) is defined as follows: CI = mean no. of cells counted per high-power oil-immersion field ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effect of GM-CSF on CCR mRNA expression, we
performed RNase protection assays on PMN treated with increasing
concentrations of GM-CSF (0.01–10 ng/ml, 6 h; Fig. 1
, A and B). CCR1
and CCR3 mRNA expression was detected in cells incubated in medium
alone (6 h). Following incubation with GM-CSF, CCR1 expression was
up-regulated, with an effect being apparent at a GM-CSF concentration
of 0.01 ng/ml. In contrast, PMN treated with the same concentration
range of GM-CSF did not show an increase in CCR3 expression above
levels detected in control cells. CCR2, CCR4, and CCR5 expression were
not detected under any of the conditions tested (Fig. 1A).
Up-regulation of CCR1 expression by GM-CSF treatment (10 ng/ml)
occurred rapidly, with increases being detectable by 30 min after
addition of the stimulus (Fig. 2, A and B). Maximal CCR1 mRNA expression was
detected between 1–2 h post-GM-CSF treatment, and declined to basal
levels by 8 h. No changes in CCR1 or CCR3 expression were measured
in cells treated with medium alone over this time period. Because
IFN-
has previously been shown to induce CCR1 expression in
neutrophils (33), we also investigated whether GM-CSF and
IFN- had a synergistic effect on CCR1 expression. These two
cytokines did not synergize in inducing CCR1 expression in these cells,
instead demonstrating additive effects (data not shown).
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). CCR3
expression in control cells was variable in the four donors tested,
with an immunoreactive band detectable in one donor (Fig. 3) and
undetectable in three donors (data not shown). In all cases, treatment
with GM-CSF did not yield a detectable increase in CCR3 protein
expression, including the experiment in which control cells expressed
CCR3 (Fig. 3). Thus, we have shown that GM-CSF up-regulated CCR1 mRNA
and protein, but did not affect CCR3 mRNA or protein expression.
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Because GM-CSF selectively up-regulated the expression of CCR1, we
sought to determine whether CCR could be activated in cells treated
with GM-CSF. Because chemokine-induced calcium responses are often used
as a measure of chemokine receptor activation, we used standard
fluorometric techniques to assess chemokine-induced calcium transients
in GM-CSF-stimulated PMN. PMN cultured in GM-CSF for 16 h showed
transient intracellular calcium increases in response to three known
CCR1 agonists (34, 35), MIP-1, MCP-3, and RANTES (100
ng/ml), but not to eotaxin, MCP-1, MCP-4, or MIP-1 (Fig. 4). Because MIP-1, MCP-3, and RANTES
all bind to CCR1, this result correlated with GM-CSF induction of CCR1
mRNA and protein expression (
Figs. 1–3
). Although constitutive CCR3
expression was observed in resting and GM-CSF-stimulated PMN (Fig. 1),
the CCR3-specific ligand eotaxin did not stimulate intracellular
calcium mobilization in this assay (Fig. 4). Control PMN cultured in
medium alone for 16 h did not respond to any of the CC chemokines
tested (Fig. 4, untreated). The effect of GM-CSF on calcium responses
was time-dependent, as cells treated for less than 16 h did not
show calcium transients in response to CC chemokines (data not
shown). MIP-1, MCP-3, and RANTES stimulated intracellular calcium
increases in a concentration-dependent fashion (100–500 ng/ml; Fig. 5), with the minimum effective
concentration being 100 ng/ml. GM-CSF treatment enhanced
IL-8-induced calcium responses, an effect probably due to priming
effects because GM-CSF has no effect on the expression of IL-8
receptors CXCR1 and CXCR2 (22).
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, MCP-3, or RANTES were activating
intracellular calcium mobilization through the same receptor, we
investigated the desensitization relationships between these three
chemokines in GM-CSF-treated PMN (Fig. 6). Pretreatment of PMN with MIP-1
(200 ng/ml) rendered the cells unresponsive to further stimulation by
MCP-3 or RANTES (200 ng/ml; Fig. 6, A and B).
Similarly, RANTES treatment (200 ng/ml) blocked responses to subsequent
stimulation with MIP-1 or MCP-3 (200 ng/ml; Fig. 6, C and
D). In contrast, pretreatment of PMN with MCP-3 (200 ng/ml)
partially blocked, but did not fully abolish, calcium responses to
MIP-1 or RANTES (200 ng/ml; Fig. 6, E and
F).
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Once we had established that functional CCR were present on the
surface of GM-CSF-stimulated PMN, we investigated whether any CC
chemokines were chemotactic for GM-CSF-treated PMN using a chemotaxis
microchamber technique. MIP-1, MCP-3, and RANTES were able to induce
migration of cells that had been incubated for 16 h with 10 ng/ml
GM-CSF, but showed no activity on cells treated with culture medium
alone (Fig. 7) or freshly isolated PMN
(data not shown). This result correlates with the RNase protection
assay results documenting GM-CSF induction of CCR1 mRNA expression
(Fig. 1 and 2), because MIP-1, MCP-3, and RANTES are CCR1 agonists.
Migration to each of these chemokines was abolished in the absence of a
concentration gradient (data not shown), indicating chemotactic
(gradient-oriented) rather than chemokinetic (increased random motion)
activity. Although constitutive CCR3 expression was observed in resting
and GM-CSF-stimulated PMN (Fig. 1), the CCR3-specific ligand eotaxin
did not induce migration in this assay (data not shown). The CC
chemokines MIP-1, MCP-1 and MCP-4 did not show any chemoattractant
activity on PMN in this assay over the concentration range used (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
MCP-3, and RANTES, while control cells did not. This is consistent with
the interpretation that resting neutrophils express a level of CCR1
that is below the threshold needed to make the cells functionally
responsive to the corresponding ligands, with a moderate increase being
enough to push this level above the threshold. A threshold model of
chemoattractant receptor activation in neutrophils that supports this
interpretation has been previously established (45).
Neutrophil recruitment is a critical component of the immune response
to many bacterial and fungal pathogens (15, 46).
Recruitment of these cells is necessary for efficient clearing of the
invading pathogen, with a strong correlation between reduced neutrophil
responses and increased disease severity. Several studies using
neutralizing anti-MIP-1 Abs have demonstrated that MIP-1 is
necessary for in vivo neutrophil recruitment in a variety of murine
models of inflammation, such as experimental bacterial meningitis
(46), immune complex-induced lung injury
(13), mycobacterial infection (47), and
endotoxemia-associated lung injury (14). In addition,
targeted disruption of the murine gene encoding CCR1, a receptor known
to bind MIP-1, has been shown to result in deficient neutrophil
responses and increased morbidity in infectious aspergillosis
(15), indicating that MIP-1-mediated neutrophil
recruitment is an important component of the immune response. However,
it is unclear whether MIP-1 exerts direct effects on PMN, or works
indirectly via production of classical neutrophil chemoattractants,
such as CXC-type chemokines, from mononuclear or stromal cells. Murine
PMN isolated from peripheral blood and inflammatory sites respond to
MIP-1 with chemotaxis and intracellular calcium transients in vitro
(15), suggesting that direct effects are
possible in vivo.
Although CCR1 has been shown to be a functional receptor on mouse PMN,
its function on human PMN is not as clearly understood. The literature
contains conflicting evidence regarding the existence of CCR on human
PMN. CCR1 mRNA expression has been documented in resting human PMN
(34, 48), although surface expression has been
undetectable by flow cytometric staining (49). A few
groups have reported that PMN do possess binding sites for MIP-1
(10, 12), MCP-3 (10), and leukotactin
(12), all of which bind to CCR1 (10, 12),
although McColl et al. (11) were unable to detect MIP-1
binding to PMN using similar methods and conditions. Although MIP-1
does not show chemotactic activity for resting human PMN
(10, 11, 12), it has been reported that other CCR1 agonists
such as leukotactin (12), MCP-3 (10), CK8,
and CK8–1 (32) are chemotactic for this cell type.
However, other groups have not been able to demonstrate some of these
results (33, 50). It is likely that resting PMN possess
low levels of surface CCR1 that are insufficient for full activation of
signaling pathways.
The conflicting results observed between studies on the role of CC chemokines in the chemotaxis of PMN may be explained by at least two different hypotheses. Firstly, it is possible that fundamental differences in the structure and function of the chemokine system between mouse and human may exist. For example, while the mouse has an orthologue of the human IL-8 receptor subtype CXCR2 (51), murine counterparts of IL-8 and the other human IL-8 receptor CXCR1 have not been identified. Secondly, differences in activation state of cells may affect the functional status of CCR on PMN, either by increasing the level of surface expression, or increasing availability of downstream signaling molecules. This would be consistent with findings in other cell types such as T lymphocytes, where chemokine receptor signaling in specific T cell subsets is regulated both at the level of receptor expression and receptor signaling (52). In this case, in vivo activation of PMN by factors such as cytokines or adhesion molecules might allow them to broaden their range of chemokine responsiveness to include those of the CC family.
The findings presented in this paper showing that PMN activated by
GM-CSF up-regulate CCR1 expression suggest that cellular activation can
broaden the spectrum of CC chemokine activity in vivo to include PMN.
This model has been introduced in a recent study by Bonecchi et al.
(33) demonstrating that CCR1 and CCR3 can be up-regulated
on PMN by the pro-inflammatory cytokine -IFN. Our results differ
from the Bonecchi study in that GM-CSF specifically up-regulated CCR1
expression without modulating CCR3 expression. Together these studies
show that different cytokines have specific effects on chemokine
receptor expression patterns and may be able to fine-tune the chemokine
responsiveness of activated PMN. Our investigations together with the
Bonecchi study also suggest that the apparent contradictions present in
the literature concerning CCR expression on PMN may be due in part to
different cellular activation states brought about by differences in
cell handling and isolation procedures or donor variability.
In the present study, the CC chemokines MIP-1, RANTES, and MCP-3
induced chemotaxis in GM-CSF-treated PMN. Furthermore, our
investigations have demonstrated that MIP-1, RANTES, and MCP-3 could
induce calcium mobilization in GM-CSF-stimulated PMN. Pretreatment of
PMN with MIP-1 completely abolished responses to both RANTES and
MCP-3, while pretreatment with RANTES also desensitized the cells to
MIP-1 and MCP-3. Such a reciprocal desensitization relationship
suggests that these chemokines are all working through the same
receptor, CCR1. However, MCP-3 treatment could not fully desensitize
the calcium response to MIP-1 and RANTES. One explanation for this
is that MCP-3 might not efficiently cause receptor internalization,
which is the main mechanism for chemokine receptor desensitization
(36). It has been documented that MCP-3 can bind to CCR5
but is a poor stimulus for internalization of that receptor
(53); it is possible that this characteristic applies to
its binding to CCR1 as well.
Our data is consistent with GM-CSF up-regulation of surface CCR1, rendering the cells responsive to CCR1 ligands in assays of chemotaxis and calcium flux. This work suggests that activation of PMN can widen the spectrum of CC chemokines to include this cell type, which is traditionally thought of as responding to CXC chemokines alone. This mechanism might be functional within inflamed tissues, where GM-CSF is made locally by stromal cells and mononuclear phagocytes. Alternatively, systemic activation of endothelial cells has been shown to cause GM-CSF-dependent intravascular activation of PMN (40). It has long been known that in systemic inflammatory states, peripheral PMN lose their responsiveness to CXC chemokines (54, 55). Our work poses the question of whether these cells might gain responsiveness to CC chemokines, providing a possible mechanism for the PMN-mediated tissue damage associated with a variety of inflammatory conditions (55, 56). Such scenarios of local and systemic PMN activation and responsiveness to CC chemokines lead to new and interesting questions on PMN participation in cytokine networks and in the pathogenesis of inflammatory lesions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven L. Kunkel, Department of Pathology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein.
Received for publication December 21, 1999. Accepted for publication October 12, 2000.
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S. J. Campbell, V. H. Perry, F. J. Pitossi, A. G. Butchart, M. Chertoff, S. Waters, R. Dempster, and D. C. Anthony Central Nervous System Injury Triggers Hepatic CC and CXC Chemokine Expression that Is Associated with Leukocyte Mobilization and Recruitment to Both the Central Nervous System and the Liver Am. J. Pathol., May 1, 2005; 166(5): 1487 - 1497. [Abstract] [Full Text] [PDF]
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C. L. Speyer, H. Gao, N. J. Rancilio, T. A. Neff, G. B. Huffnagle, J. V. Sarma, and P. A. Ward Novel Chemokine Responsiveness and Mobilization of Neutrophils during Sepsis Am. J. Pathol., December 1, 2004; 165(6): 2187 - 2196. [Abstract] [Full Text] [PDF]
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J. Gomez-Cambronero, J. Horn, C. C. Paul, and M. A. Baumann Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway J. Immunol., December 15, 2003; 171(12): 6846 - 6855. [Abstract] [Full Text] [PDF]
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C. L. Speyer, T. A. Neff, R. L. Warner, R.-F. Guo, J. V. Sarma, N. C. Riedemann, M. E. Murphy, H. S. Murphy, and P. A. Ward Regulatory Effects of iNOS on Acute Lung Inflammatory Responses in Mice Am. J. Pathol., December 1, 2003; 163(6): 2319 - 2328. [Abstract] [Full Text]
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 X. Zeng, T. A. Moore, M. W. Newstead, R. Hernandez-Alcoceba, W. C. Tsai, and T. J. Standiford Intrapulmonary Expression of Macrophage Inflammatory Protein 1{alpha} (CCL3) Induces Neutrophil and NK Cell Accumulation and Stimulates Innate Immunity in Murine Bacterial Pneumonia Infect. Immun., March 1, 2003; 71(3): 1306 - 1315. [Abstract] [Full Text] [PDF]
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 D. K. Bhalla, P. G. Reinhart, C. Bai, and S. K. Gupta Amelioration of Ozone-Induced Lung Injury by Anti-Tumor Necrosis Factor-{alpha} Toxicol. Sci., October 1, 2002; 69(2): 400 - 408. [Abstract] [Full Text] [PDF]
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) or GM-CSF (10 ng/ml; 
) for 16 h.
Results are expressed as mean chemotactic index ± SEM of three
replicate measurements from a single experiment and is representative
of three to five separate experiments.