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1
Departments of
*
Microbiology and Immunology,
Pathology, and
Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mediated dose-dependent migration of human cord blood-derived
mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1
did not induce mast cell degranulation, it selectively stimulated
production of the neutrophil chemoattractant IL-8 without affecting
TNF-, IL-1ß, IL-6, GM-CSF, IFN-
, or RANTES production,
providing further evidence of the selective modulation of mast cell
function by this chemokine. These findings provide a novel,
SDF-1-dependent mechanism for mast cell transendothelial migration and
functional regulation, which may have important implications for the
local regulation of mast cells in disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell migration requires the presence of chemotactic factors and the
expression of specific cell surface receptors on the target cells. A
growing body of evidence shows that a family of structurally related
chemotactic proteins, namely chemokines, plays an essential role in the
selective recruitment of inflammatory cells (8).
Chemokines are small m.w. proteins characterized by the presence of a
conserved motif containing four cysteine residues. In chemokines,
the two cysteine residues are separated by a single amino acid, with
their corresponding receptors designated CXC receptors (CXCR). Five
types of CXCR have been identified, namely, CXCR1, -R2, -R3, -R4, and
-R5 (8, 9, 10). In ß chemokines, the two cysteine residues
are adjacent, with their corresponding receptors designated CC
receptors (CCR), including CCR1 through CCR9 (8, 9, 10). In
contrast to other cell types such as lymphocytes, little is known about
chemokine receptor expression on mast cells and the factors that
mediate mast cell migration. In particular, factors that can induce the
migration of mast cells across an endothelial monolayer have not been
previously described, although this process is clearly essential for
blood-borne mast cell precursors to reach tissue. Several and ß
chemokines, including IL-8, monocyte chemoattractant protein-1
(MCP-1),3 RANTES,
platelet factor 4, and MIP-1, and some angiogenic factors
(11) and growth factors, such as stem cell factor (SCF)
(12), TGF-ß (13, 14), and IL-3
(15), have been reported to exert chemotactic effects on
rodent mast cells (16). Many of these, including MIP-1,
MIP-1ß, MCP-1, MCP-2, MCP-3, IL-3, TGF-ß, and nerve growth factor,
have been reported to be ineffective on human mast cells (17, 18). Some chemokines/cytokines, such as IL-8, SCF, and RANTES,
have been shown to exert chemotactic activity on human mast cells in
some studies (17, 19, 20), but not in others (17, 18). Moreover, all in vitro studies to date were designed to
determine mast cell migration on extracellular matrix proteins, such as
laminin, fibronectin, vitronectin, or collagens. Thus, the factors
potentially driving transendothelial migration of developing human mast
cells and the receptor(s) systems used remain to be determined.
Mast cells have been convincingly implicated in a number of allergic
inflammatory diseases as well as in host defense against pathogens
through recruitment of other inflammatory cells, such as neutrophils
(1, 21, 22, 23). IL-8, a potent neutrophil chemoattractant and
activator, can be produced by human mast cells (24, 25).
However, little is known about the mechanisms that regulate IL-8
production by mast cells. A number of well-known mast cell
secretagogues, such as anti-IgE, substance P, and Con A, cannot
elicit IL-8 production from mast cells (24). Moreover,
several stimuli, including IL-1, IL-1ß, and LPS, that are known to
cause IL-8 secretion from other cells, such as monocytes or
fibroblasts, did not induce IL-8 release from mast cells
(24). Thus, it appears that mast cells possess unique
mechanisms to regulate IL-8 production. Stimuli that have been shown to
induce IL-8 production by mast cells include PMA, calcium ionophore
A23187, N-ethylcarboxamidoadenosine, anti-CD43 Ab, and
lymphocyte membrane preparations (24, 25, 26). No endogenous
mammalian proteins have previously been shown to elicit IL-8 production
by mast cells, although IL-4 can enhance ionomycin-induced IL-8
expression (27).
Stromal cell-derived factor-1 (SDF-1), an chemokine expressed by
stromal cells and fibroblasts (28), is a highly potent
lymphocyte chemoattractant (29). SDF-1 was recently
identified as the only known ligand for CXCR4 (30, 31).
Several cell types, including naive T lymphocytes (32, 33), B cells (32, 34), monocytes (32, 33), dendritic cells (35), megakaryocytes
(36), microglial cells, and astrocytes (37)
have been shown to express CXCR4. However, this receptor is only weakly
present on NK cells (33), and it has been suggested that
it is not expressed on memory T cells or a subpopulation of germinal
center B cells (32). SDF-1 exerts chemotactic activity on
several cell types, including some lymphocytes, but not on others, such
as neutrophils (29) and astrocytes (37).
Thus, the expression of CXCR4 and the function of SDF-1 are not
ubiquitous, but, rather, are very selectively dependent on cell
type.
In this study we have demonstrated the expression of the SDF-1
receptor, CXCR4, on the cell surface and intracellularly in human mast
cells and reported for the first time the transendothelial migration of
human mast cells in response to SDF-1. Moreover, SDF-1 selectively
induced IL-8 production without affecting TNF-, GM-CSF, and IL-6
production or mast cell degranulation. Our results suggest a role for
SDF-1 in mast cell recruitment and function under physiological or
pathological conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SDF-1 was purchased from PeproTech (Rocky Hill, NJ).
According to the manufacturer, it is expressed in Escherichia
coli and is >98% pure by SDS-PAGE and HPLC analyses.
The endotoxin level is <0.1 ng/µg (1 EU/µg) of SDF-1. Mouse
anti-human CXCR4 (fusin) Ab (12G5, IgG2a) and mouse anti-human
CD13 mAb (clone WM15, IgG1) were purchased from PharMingen (San Diego,
CA). Goat anti-CXCR4 Ab (G-19) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse anti-human c-Kit mAb
(clone K44.2, IgG1) was purchased from Sigma (St. Louis,
MO).
Mast cells
Highly purified cord blood-derived mast cells (CBMC) were obtained by long term culture of cord blood progenitor cells as previously described (38). Briefly, heparinized cord blood, obtained after informed consent of the patients, was centrifuged over a Ficoll separating solution (Seromed, Berlin, Germany). Light density cells, including the progenitors, were cultured at 37°C in a humidified atmosphere containing 5% CO2 at a starting density of 1 x 106 cells/ml in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, 10% FCS (all from Life Technologies, Grand Island, NY), 1% (w/v) BSA (Sigma), 50 µM 2-ME (Sigma), 100 ng/ml human recombinant SCF (a gift from Amgen, Thousand Oaks, CA), and 20% CCL-204 (American Type Culture Collection, Manassas, VA) normal human skin fibroblast supernatant as a source of IL-6. The medium was renewed every 7 days. The percentage of mast cells in the cultures was assessed by toluidine blue staining (pH 1.0) of cytocentrifuged samples. After >8 wk in culture, mature mast cells were identified by their morphological features and the presence of metachromatic granules and used in our study.
The human mast cell line HMC-1 5C6 (39), a more differentiated subclone from its parental line, HMC-1 was grown in Iscove’s medium (Life Technologies) supplemented with 10% FBS (Life Technologies). After confluent growth, the adherent cells were harvested by subtle pipetting.
The human basophilic cell line, KU812, was maintained in RPMI 1640 (Life Technologies), supplemented with 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. For further differentiation, KU-812 cells were cultured with SCF (80 ng/ml) and IL-6 (50 ng/ml) for 7 days.
Mouse BMMC from male C57BL/6 mice and peritoneal mast cells (PMC) from Lewis rats were obtained as previously described (40, 41).
For intracellular staining, mast cells were fixed, permeabilized, and stained. Briefly, cells were washed with cold PBS and fixed with 4% paraformaldehyde for 5 min. After washing, cells were resuspended in 10% DMSO in PBS and stored at -80°C. Thawed cells were washed and incubated with 0.1% saponin and 3% BSA in PBS for 1 h at room temperature. After washing, cells were stained with Abs.
Flow cytometric analysis
In 96-well U-bottom plates, cells (5 x 105 cells/test) were incubated with the primary Ab in immunofluorescence (IF) buffer (PBS, 1% BSA, and 0.2% sodium azide) for 1 h at 4°C. After washing cells were further incubated for 45 min with secondary Ab, FITC-conjugated goat anti-mouse IgG (Zymed, San Francisco, CA), or FITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology). For double staining, cells were stained with mouse anti-human c-Kit Ab and goat anti-CXCR4 Ab for 1 h at 4°C, followed by staining with swine anti-goat IgG-PE and sheep anti-mouse IgG-FITC for 45 min. Cells were washed three times (with IF buffer) and resuspended in 400 µl of 1% formalin (in IF buffer), and 10,000 cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA). The results with specific Abs were compared with those using control Abs.
RT-PCR
Total RNA was extracted from HMC-1 5C6, KU812, mouse BMMC, or rat PMC using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA from rat PMC were treated with heparinase to remove contaminating heparin (41). RT using MMLV transcriptase (Life Technologies) and PCR using Taq DNA polymerase (Life Technologies) were performed according to the manufacturer’s protocols with some modifications (41). The primers used were 1) human CXCR4: sense, 5'-TAA CTA CAC CGA GGA AAT GGG C-3'; antisense, 5'-ACC ATG ATG TGC TGA AAC TGG-3'; 2) mouse CXCR4: sense, 5'-ACT ACT CTG AAG AAG TGG G-3'; antisense, 5'-ATG AGA ACG CTG CTG TAG AGG-3'; and 3) rat CXCR4: sense, 5'-CAC TTC GGA TAA CTA CTC C-3'; antisense, 5'-TAA CAG GAC AGG ATG ACG ATG C-3'. The PCR products for human, mouse, and rat CXCR4 were 588, 346, and 636 bp, respectively. Thirty-five cycles were used (95°C for 45 s, 56°C for 45 s, and 72°C for 2 min). Products were run on a 2% agarose gel and stained with ethidium bromide.
Intracellular Ca2+ measurement
Mast cells (5 x 106 cells/ml) were incubated in PBS for 30 min with 2 µM fluo-4/AM (Molecular Probes, Eugene, OR). After washing, mast cells were resuspended in PBS with 1.5 mM CaCl2 at a concentration of 1 x 106 cells/ml. Fluorescence was measured by placing 2 ml of the mast cell suspension in a 37°C thermostated quartz cuvette with magnetic stirring in a RF-1501 spectrofluorophotometer (Shimadzu, Tokyo, Japan). Fluorescence was recorded at 520 nm after excitation at 485 nm.
Confocal microscopy imaging of CXCR4
Cells (5 x 105 cells/test) were incubated with mouse anti-human CXCR4 mAb 12G5 or mouse IgG2a for 1 h at 4°C. After washing, cells were further incubated with goat anti-mouse IgG-FITC for 45 min. Cells were washed three times and resuspended in 1% formalin. Cytospins of FITC-labeled mast cells were made by vortexing slides in a Cytospin 3 (Shandon, Cheshire, U.K.) at 600 rpm for 3 min. Anti-bleaching solution (10 mM propyl gallate (Sigma) and 8.1 M glycerol in Tris-buffered saline) was dropped onto slides before coverslip attachment. Cells were examined with a Zeiss LSM410 confocal laser scanning microscope (Jena, Germany).
ß-Hexosaminidase (ß-Hex) assay
Human CBMC were preincubated with SDF-1 at a concentration of
1000 ng/ml for 30 min and was further incubated with ionophore A23187
(1 µM) or PMA (100 nM) for 20 min. Rat PMC were incubated
with SDF-1 or ionophore A23187 for 20 min. ß-Hex was measured in
both supernatant and pellet fractions using a previously reported
method (42). Briefly, 50 µl of each sample was incubated
with 50 µl of 1 mM
-nitrophenyl-N-acetyl-ß-D-glucosaminide
(Sigma) dissolved in 0.1 M citrate buffer, pH 5, in a 96-well
microtiter plate at 37°C for 1 h. The reaction was stopped with
200 µl/well of 0.1 M carbonate buffer, pH 10.5. The plate was read at
405 nm in an ELISA reader.
Bioassay for TNF- and IL-6
TNF- and IL-6 in cell-free supernatants was tested as
cytotoxicity of WEHI 164.13 (TNF-) or bioactivity on B-9 hybridoma
proliferation (IL-6) using MTT assay as previously described (41, 43). Human recombinant TNF- and IL-6 (Genzyme, Cambridge, MA)
were used as standards. The sensitivity of these assays was 10 U/ml for
IL-6 and 1 pg/ml for TNF-, respectively. These assay systems do not
detect other known mast cell-derived cytokines under the conditions
employed here. One unit of IL-6 is equivalent to 
0.45 pg.
IL-8, IL-1ß, GM-CSF, IFN-, and RANTES ELISAs
Human IL-8, GM-CSF, and IL-1ß levels in supernatants or
pellets were measured using an in-house ELISA. Briefly, 96-well plates
were coated with anti-human IL-8 (R&D Systems, Minneapolis, MN),
anti-human GM-CSF Ab (Genzyme), or anti-human IL-1ß (Endogen,
Woburn, MA) at 1 µg/ml for 16–20 h at 4°C. Nonspecific
binding to the plates was blocked using a 1% BSA/0.1% Tween-20
solution in PBS for 1 h at 37°C. A total of 50 µl/well of IL-8
(human rIL-8; R&D Systems), GM-CSF (human rGM-CSF; R&D Systems), or
IL-1ß standard (human rIL-1ß; Endogen), and samples were added to
the plate and incubated for 18–20 h at 4°C. Biotinylated
anti-human IL-8 (R&D Systems), anti-human GM-CSF (Endogen), or
anti-human IL-1ß (Endogen; 0.2 µg/ml) was added to each well
and incubated for 1 h at 37°C. After washing, 50 µl/well of a
1/2000 dilution of streptavidin-alkaline phosphatase (Life
Technologies) was added according to the manufacturer’s instructions.
The minimal detectable dose was 3 pg/ml for IL-8, GM-CSF, and IL-1ß
using this system. Human IFN- and RANTES were measured using ELISA
kits purchased from Endogen according to the manufacturer’s
instructions.
Isolation and culture of human endothelial cells
HUVEC were isolated and cultured as described by Jaffe (44), and HUVEC monolayers on filters were grown as described previously (45). Briefly, endothelial cells isolated from umbilical cords by collagenase treatment were grown in RPMI 1640 (Sigma) containing 2 mM L-glutamine, 2-ME, sodium pyruvate, penicillin G/streptomycin, 20% FBS (HyClone), 25 µg/ml endothelial cell growth factor (Collaborative Research, Lexington, MA), and 22.5 µg/ml heparin (Sigma) in gelatin-coated flasks (Nunc, Naperville, IL). The HUVEC were detached with 0.025% trypsin/0.01% EDTA and cultured on the Transwell filters (Corning Costar, Acton, MA). The filters were precoated with 0.01% gelatin (37°C overnight) followed by 3 µg of human fibronectin (Collaborative Research) in 45 µl of water at 37°C for 2 h. The HUVEC (1.5 x 104 cells in 0.1 ml of complete medium) from first or second passage were added above the filter, and 0.6 ml of medium was added to the lower compartment beneath the filter. The cells became confluent and formed a tight monolayer in 5–6 days, with a permeability of <1.5% as assessed by [125I]HSA diffusion (45).
Mast cell migration across endothelial monolayers
The transendothelial migration of human mast cells was performed
using HUVEC monolayers. Briefly, human mast cells were labeled with
51Cr sodium chromate (25 µCi/ml; Amersham,
Oakville, Canada) by incubation for 45 min at 37°C. Medium (0.1 ml)
containing 1–2 x 105 Cr-labeled human mast
cells were added above the HUVEC monolayers on the filters, and the
Transwells were placed into 0.6 ml of RPMI 1640 plus 0.5% HSA in a
24-well plate. SDF-1 was added at varying concentrations to the
lower chamber as chemotactic stimulus. After 4-h incubation, the
migration was stopped. The undersurface of the filter was rinsed into
the lower compartment and swabbed with a cotton swab soaked in ice-cold
PBS/0.2% EDTA. The cells that migrated into the lower compartment were
collected and combined with the contents of the swab for
51Cr analysis to determine the total
51Cr mast cells migrating through the filter,
referred to as migrated cells. All the treatment conditions were
performed in duplicate or triplicate.
MTT assay for mast cell proliferation
HMC-1 cells (5 x 105 cells/ml) were
treated with SDF-1 (0.1, 1, 10, 100, and 1000 ng/ml) for 24 h
and further incubated with MTT (0.5 mg/ml) for 2 h.
Isopropanol-HCl was used to dissolve the purple formazan precipitates.
The OD was measured at 570 nm.
Statistical analysis
ANOVA and paired Student’s t test were used for statistical evaluation of data. Results were considered significant when p < 0.05. Throughout the text, data are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether mast cells express mRNA for CXCR4, RNA from
various mast cell populations, including the human mast cell line HMC-1
5C6, the human basophil/mast cell line KU812, freshly isolated rat PMC,
or cultured mouse BMMC, were reverse transcribed. After establishing a
positive PCR product for ß-actin (data not shown). cDNA was subjected
to PCR amplification with specific primers for human, rat, or mouse
CXCR4, respectively. As shown in Fig. 1
,
positive PCR products were detected in HMC-1 and KU812 (588 bp), rat
PMC (636 bp), and mouse BMMC (346 bp). No PCR products were detected
when reverse transcriptase was omitted (data not shown).
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). A large proportion of CBMC
stained positively for both the mast cell marker c-Kit or CD13 and
CXCR4. Moreover, >90% of the human mast cell line HMC-1 and KU812
were positive with Ab G-19. No Ab cross-reactivities were observed when
using nonimmune goat IgG with mouse anti-human c-Kit or mouse
anti-human CD13 and goat anti-CXCR4 with mouse IgG as controls
(not shown).
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) stained positively (>90%) for
G-19 (CXCR4). Jurkat cells were used as a positive control and stained
positively (>90%; data not shown). Intracellular and cell surface expression of CXCR4 on human mast cells
To further confirm that mast cells express CXCR4 protein and
determine the intracellular and cell surface expression, another
anti-CXCR4 Ab 12G5, which reacts with the first and second
extracellular loops of CXCR4, was used to stain permeabilized human
mast cells. HMC-1 cells harvested from culture were fixed by 4%
paraformaldehyde and permeabilized with saponin. These cells were then
stained with anti-CXCR4 Ab 12G5 and examined by confocal
microscopy. Both cell surface and intracellular compartments stained
for CXCR4 (Fig. 3A) compared
with the isotype-matched control Ab (Fig. 3B).
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, D and E), suggesting abundant
intracellular and cell surface CXCR4 protein. Permeabilization
treatment also increased the background fluorescence staining.
Given that CXCR-4 expression on T cells or dendritic cells can be
up-regulated by IL-4 (46, 47) or TGF-ß1
(47) and down-regulated by IFN- (47), we
attempted to test whether these cytokines could modulate CXCR-4
expression on mast cells. HMC-1 cells were treated with IL-10, IL-4,
IFN-, IL-6, or GM-CSF (0.1–1 µg/ml) for 6, 24, or 48 h and
tested for CXCR-4 expression using Abs 12G5 and G-19 in unpermeabilized
or permeabilized conditions. Interestingly, expression of CXCR-4 by
HMC-1 cells was not modified by these cytokines after 6-, 24-, or 48-h
incubation (data not shown).
SDF-1 induces calcium responses in human mast cells
To test whether CXCR4 protein is functional in mast cells, we
measured intracellular calcium levels after stimulation with its
natural ligand, SDF-1 (30). Treatment with SDF-1
increased intracellular calcium levels in both CBMC and HMC-1 (Fig. 4). The rapid calcium flux was noted
within 5 s. Interestingly, no calcium response was observed in
KU812 cells after SDF-1 stimulation, while a significant calcium
flux in response to C5a could be observed in these cells (data not
shown). As noted in Fig. 4C, stimulation with 500 ng/ml
SDF-1 completely desensitized HMC-1 cells for the subsequent
challenge with the same dose of SDF-1, whereas SDF-1 did not
cross-desensitize HMC-1 cells for the calcium response to C5a.
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induces migration of human mast cells
The capacity of SDF-1 to induce the migration of human mast
cells through confluent monolayers of HUVEC was examined in Transwell
plates. Mast cells transmigrated through endothelium were harvested and
quantified. Human CBMC from four different donors showed a
concentration-dependent transendothelial migration in response to
SDF-1 (Fig. 5). There was a
considerable variation in the degree of transendothelial migration
between mast cells from different donors, but in each case SDF-1
stimulated a significant increase in mast cell transendothelial
migration. HMC-1 cells also demonstrated transendothelial migration in
response to SDF-1 (Fig. 5). We used anti-CXCR4 Ab 12G5 to
confirm that the transendothelial migration is specifically mediated by
SDF-1. CBMC from two donors were pretreated with Ab 12G5 (13
µg/ml) for 15 min and subjected for transendothelial migration.
Pretreatment with Ab 12G5 significantly blocked SDF-1-induced CBMC
transmigration (77.0 ± 11.7% inhibition). In contrast, Ab TA3
(anti-LFA-1) and 6F8.5D12 (anti-VLA-4) had no significant
effect. SDF-1 had no effect on the permeability of the endothelial
monolayers over the course of the assay as determined by
[125I]HSA studies (45).
Endothelial permeability values ranged between 1.3 and 1.9% in these
transmigration experiments. Treatment of endothelial monolayers with
500 ng/ml SDF over the time course of the assay had no significant
effect on the assayed permeability of
[125I]HSA.
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does not mediate mast cell degranulation, but selectively
stimulates IL-8 production
We examined whether SDF-1 could induce mast cell degranulation or
modulate mast cell degranulation in response to other stimuli. Human
CBMC were incubated with SDF-1 (1 µg/ml) for 30 min and further
incubated for 20 min in the presence or the absence of the calcium
ionophore A23187 (1 µM) or PMA (100 nM). ß-Hex secretion was
measured. Treatment of human CBMC with SDF-1 did not induce ß-Hex
secretion directly or modulate ionophore A23187- or PMA-mediated
ß-Hex release (Fig. 6). Because of the
high degree of homogeneity between human and rodent SDF-1, human SDF-1
has been successfully used in rodents (37). Thus, we
tested the efficacy of human SDF-1 on degranulation of another mast
cell population widely used for degranulation studies, rat PMC. After
incubation of rat PMC with SDF-1 (1 µg/ml) for 30 min, ß-Hex
release was determined. SDF-1 did not induce ß-Hex secretion from
rat PMC (3.32 ± 1.06% release) compared with medium alone
(2.71 ± 0.75% release). As a positive control, calcium ionophore
A23187 (0.5 µM) stimulated a significant ß-Hex release from rat PMC
(47.83 ± 0.81% release; n = 3).
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affects mast cell cytokine
expression. After 24-h incubation of HMC-1 (200 µl of
5x105 cells/ml) with SDF-1 (50 or 500
µg/ml), supernatants were collected for detection of IL-8, TNF-,
GM-CSF, IL-6, IL-1ß, IFN-, and RANTES. HMC-1 cells secreted
significant amounts of these cytokines after stimulation with calcium
ionophore and PMA. However, no effects of SDF-1 treatment on
TNF-, GM-CSF, IL-6, IL-1ß, IFN-, or RANTES production were
observed (Table I). Interestingly, IL-8
production by HMC-1 was selectively stimulated by SDF-1 treatment in
a time- and dose-dependent manner (Fig. 7). As a positive control, calcium
ionophore together with PMA induced significant IL-8 production at
24 h (194.0 ± 53.2 pg/ml) and 48 h (197.9 ± 88.2
pg/ml). SDF-1-induced IL-8 production was confirmed using human CBMC
from two donors, although there is a considerable variation in IL-8
response between mast cells from different donors. After a 24-h
incubation with SDF-1 (500 ng/ml), human CBMC
(1x106 cells/ml) secreted significant amounts of
IL-8 (donor 1: no treatment, 14.6 ± 4.6 pg/ml; SDF-1,
80.2 ± 55.0 pg/ml; A23187 plus PMA, 34.6 ± 19.5 pg/ml;
donor 2: no treatment, 103.6 ± 30.7 pg/ml; SDF-1, 331.6
± 204.7 pg/ml; A23187 plus PMA, 228.4 ± 5.1 pg/ml; in
duplicate).
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alters the proliferation of HMC-1 cells, which may reflect an increased
IL-8 production. The number of HMC-1 cells was measured using the MTT
assay after incubation of the cells with various concentration of SDF-1
for 24 h. No significant differences between the SDF-1-treated
groups (OD450 values: 0.1 ng/ml, 0.57 ±
0.02; 1 ng/ml, 0.59 ± 0.02; 10 ng/ml, 0.58 ± 0.02; 100
ng/ml, 0.59 ± 0.01; 1000 ng/ml, 0.6 ± 0.01) and the
untreated group (0.63 ± 0.02) could be observed
(n = 3). This is consistent with the report by Ochi et
al. (50) that SDF-1 was not mitogenic on CBMC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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effectively mediates transendothelial migration of human mast cells.
This finding suggests a mechanism, the interaction of SDF-1 and CXCR4,
by which mast cells may be recruited to local tissue during mast cell
development or inflammation. This observation that SDF-1 can induce
transendothelial migration of human mast cells is in contrast to the
finding that SCF, which has been described to be a potent mast cell
chemoattractant, does not induce mast cell transendothelial migration
in our system. The requirements for transendothelial migration may be
much more stringent than those for chemotaxis alone. Despite well-recognized mast cell heterogeneity (48), CXCR4 was constitutively expressed in all mast cell populations tested, including human CBMC, HMC-1, SCF- and IL-6-differentiated KU812 cells, murine BMMC, and rat PMC. Interestingly, the expression of CXCR4 protein is not limited to the cell surface. Using flow cytometry and confocal microscopic analysis on permeabilized mast cells, we identified abundant intracellular CXCR4. Given that binding of SDF-1 with CXCR4 can mediate receptor internalization in T cells (49), we tested whether human mast cells express SDF-1. RT-PCR analysis demonstrated no SDF-1 message in HMC-1 cells (data not shown). Thus, it is unlikely that the intracellular CXCR4 in HMC-1 cells was internalized from the cell surface. It is possible that intracellular CXCR4 may function as a supply of the surface receptor as has been reported in lymphocytes (32).
CXCR4, like other chemokine receptors, is a member of the seven-transmembrane protein family and is the only known receptor for SDF-1. Chemokine receptors are functionally linked to phospholipases through G proteins. Receptor activation leads to a cascade of cellular activation, such as generation of inositol triphosphate and release of intracellular calcium (9). An SDF-1-mediated increase in intracellular calcium levels has been found in several cell types expressing CXCR4 (31, 35, 37). The rapid increase in intracellular calcium levels after SDF-1 stimulation indicates that this receptor on HMC-1 cells and 8-wk cultured CBMC is functional, a finding consistent with that seen in 4-wk cultured CBMC by Ochi et al. (50). In other cells, such as monocytes (31) and lymphocytes (34), binding of SDF-1 with CXCR4 mediates immediate receptor desensitization, probably through receptor phosphorylation and internalization (32, 51). Similarly, stimulation of mast cells with SDF-1 completely desensitized them for a second challenge with SDF-1, while these cells retained their responsiveness to C5a. These findings suggest that CXCR4 and C5a receptor (CD88) on mast cells function independently. The lack of cross-desensitization also suggests that mast cells responding to SDF-1 in vivo might retain their ability to be regulated by other inflammatory mediators using similar receptor systems.
Recently, Ochi et al. (50) used modified protocols to generate human CBMC and reported that CXCR4 was found in cells after 4 wk culture (containing a mixture of mast cells, mast cell precursors, and other cell types). However, these researchers could not detect CXCR-4 protein and mRNA in CBMC after 9 wk of culture. Whether this down-regulation of CXCR-4 on their CBMC preparations is due to the conditions of cell culture is unclear. However, we have consistently observed CXCR-4 expression on a wide variety of mast cell types in several species, including "mature" ex vivo mast cells from the rat peritoneum.
We and others have demonstrated that mast cells possess distinct
mechanisms to regulate degranulation and cytokine production (43, 52). Having established that SDF-1 can induce the
transendothelial migration of mast cells, we also tested the effects of
SDF-1 on mast cell degranulation and cytokine production. SDF-1
by itself did not induce human mast cell degranulation, nor did it
modulate calcium ionophore A23187- or PMA-mediated mast cell
degranulation. In addition, no effects of SDF-1 on rat PMC
degranulation could be observed in our study. Similarly, Hartmann et
al. (53) reported that several chemokines, including
RANTES, MCP-1, MCP-2, MCP-3, MIP-1, and MIP-1ß, could not elicit
human mast cell degranulation. In mice, although a number of chemokines
exhibited various chemotactic effects on mast cells, they did not
induce mast cell degranulation (16). Moreover, an in vivo
study in mice also demonstrated that mast cells in the lymph node after
migration from skin exhibited no evidence of degranulation
(54). Thus, it appears that distinct signals for mast cell
migration and degranulation exist, and that CXCR4/SDF-1 interaction is
a further example of this dichotomy of function.
The mechanisms by which mast cell numbers are increased at sites of
infection and inflammation are poorly understood. A number of
chemokines and other inflammatory mediators have been demonstrated to
induce chemotaxis of mast cells on extracellular matrix proteins in
vitro (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50). However, mechanisms by which mast
cells might undergo the much more complex, multistep process of
transendothelial migration across vascular endothelium have not been
previously investigated. SDF-1-mediated mast cell transendothelial
migration provides a novel model to study the mechanisms of human mast
cell migration relevant to inflammatory processes. No prior activation
of the HUVEC monolayers was required for SDF-1 to induce mast cell
transmigration. The observed migration of mast cells across the HUVEC
was not the result of a breakdown of barrier function of the
endothelium, because neither mast cell migration nor SDF-1 (500
ng/ml) incubation induced elevations in
[125I]HSA permeability, which ranged from
1.3–1.9%.
Mast cells are known to be critical for a number of physiological and
pathological events, including acute inflammation such as in type 1
hypersensitivity, chronic inflammation such as in asthma, wound
healing, and host defense against a number of pathogens (1, 23). Much of the mast cell’s role in these processes is
thought to be mediated by secretion of its cytokine mediators, such as
TNF-, GM-CSF, IL-6, and IL-8. The diverse roles of mast cells in
these events depend on the selective expression and secretion of
specific mast cell mediators. The selective stimulation of IL-8
production induced by SDF-1 without affecting the expression of
other cytokines/chemokines (RANTES, IL-1ß, IL-6, GM-CSF, IFN-, and
TNF-) is intriguing. IL-8 is a potent neutrophil chemoattractant and
activator (55). Mast cell-dependent neutrophil recruitment
has been shown to be critical in a number of events, such as bacterial
infection (56), IgE-mediated responses in the skin and
gastric mucosa (21, 22), and immune complex-mediated
injury (57). Given the significant interactions between
mast cells and neutrophils together with the fact that SDF-1 could be
induced by infection (58), it is likely that mast cells
recruited by SDF-1 as well as resident mast cells further amplify
immune responses by attracting and activating neutrophils through
selective secretion of IL-8. Such IL-8 production might be particularly
important in situations where mast cell degranulation products have
been able to activate local endothelium and further enhance cell
recruitment. Using the mast cell-deficient W/Wv
animal model, Zhang et al. (59) have demonstrated that
IL-8-related molecules in the context of other mast cell-derived
mediators are essential for neutrophil recruitment. Moreover, IL-4,
although unable to induce IL-8 production by itself, is able to prime
human mast cells to express IL-8 after activation (27),
suggesting further potential enhancement of IL-8 production in the
context of Th-2-associated inflammation.
In summary, we have demonstrated that human HMC-1 and CBMC and rodent
cultured and ex vivo mast cells express mRNA and protein for the
chemokine receptor CXCR4. SDF-1, the ligand for CXCR4, could stimulate
transendothelial migration of human mast cells without inducing mast
cell degranulation. Moreover, SDF-1 selectively stimulated the
production of IL-8, but not other cytokines, such as IL-6, GM-CSF,
TNF-, IL-1ß, IFN-, or RANTES. Although great caution should be
exercised when extrapolating from in vitro studies to in vivo, our
findings have identified a new, potentially important mechanism of
human mast cell migration and activation. It may be of particular
interest to evaluate the roles of SDF-1 and CXCR4 in chronic
inflammatory diseases or infection where mast cells and neutrophils are
significantly increased in number.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. S. Marshall, Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; SDF-1, stromal cell-derived factor-1; BMMC, bone marrow-derived mast cells; CXCR4, CXC chemokine receptor-4; CBMC, human cord blood-derived mast cells; SCF, stem cell factor; PMC, peritoneal mast cells; IF, immunofluorescence; HMC-1, human mast cell line-1; HSA, human serum albumin; ß-Hex, ß-hexosaminidase.
Received for publication November 3, 1999. Accepted for publication April 13, 2000.
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