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Regular Articles |
From the Department of Physiopathology, Endocrinology
Unit*
and Department of Internal Medicine, Section of
Clinical Immunology, Allergy, and Respiratory
Diseases,
University of Florence, Florence,
Italy; and the Division of Clinical Immunology and
Allergy,
University of Naples Federico II,
Naples, Italy
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and ß chemokines, which contain four cysteines, appear to be the
largest families. In the -chemokines, one amino acid separates the
first two cysteines residues (CXC), whereas in the ß-chemokines, the
first two cysteine residues are adjacent to each other (CC). The
-chemokines containing the sequence glutamic acid-leucine-arginine
near the N terminal (preceding the CXC sequence) are chemotactic for
neutrophils; those not containing the sequence act on lymphocytes. The
ß-chemokines, in general, do not act on neutrophils but attract
monocytes, eosinophils, basophils, and lymphocytes with variable
selectivity.1,3 Chemokines induce cell migration and activation by binding to specific G protein-coupled cell-surface CXCR and CCR receptors on target cells.1,3 Four human CXC chemokine receptors, CXCR1 through CXCR4, and nine human CC chemokine receptors, CCR1 through CCR9, have been identified. Chemokine receptors are expressed on different types of leukocytes, some being restricted to certain cells, others being more widely expressed not only on leukocytes but also on nonhemopoietic cells. Although most chemokine receptors bind more than one chemokine, CC receptors bind only CC chemokines and CXC receptors bind only CXC chemokines.1,3 The chemokine receptor CCR3, which binds eotaxin, RANTES, monocyte chemotactic protein (MCP)-3, MCP-4, and some other chemokines, is expressed in human eosinophils,4-6 basophils,7 and type 2 T helper (Th2) cells.8,9
In this study, by using immunohistochemistry and flow cytometry, we did not detect CCR3 expression on human Th2 cells in vitro or in vivo, whereas we found that CCR3 is expressed by basophils and, at the tissue level, by eosinophils. In addition, we demonstrate CCR3 expression on remarkable proportions of mast cells (MC) in different human tissues and on isolated cells. Although eotaxin apparently did not induce histamine release by purified human MC, both eotaxin and the CCR3 ligand, RANTES, exerted chemotactic activity on these cells. Interestingly, CCR3 expression was virtually limited to tryptase-chymase double-positive mast cells (MCTC), suggesting that migration and persistence of this MC subset into non-inflamed and inflamed tissues may depend largely on its CCR3 expression.
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Methods |
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Anti-CCR3 monoclonal antibody (mAb) (7B11; IgG2a) was kindly
provided by Leukosite (Boston, MA); anti-human eosinophil cationic
protein (ECP; clone EG2) and anti-tryptase mAb10
by
Pharmacia & Upjohn (Uppsala, Sweden); anti-macrophage associated
antigen mannose receptor (anti-PAM-1) mAb11
by A. Mantovani
(Istituto Mario Negri, Milano, Italy); anti-Fc
RI chain mAb
(IgG1) by J. Hakimi (Hoffman-La Roche Inc., Nutley, NJ); rabbit
anti-human-Fc Ab by T. and K. Ishizaka (La Jolla, CA). Anti-CD3 mAb
was purchased from Ancell (Bayport, MN); anti-chymase mAb from Chemicon
International Inc. (Temecula, CA); eotaxin, RANTES, and MIP-1 from
PeproTech EC LTD (London, UK); irrelevant anti-E-selectin mAb from R &
D Systems (Minneapolis, MN); BSA, -chymotrypsin, piperazine-N,N'-bis
(2-ethanesulfonic acid) hyaluronidase, chymopapain, collagenase,
elastase type I, and rabbit and sheep polyclonal nonimmune IgG from
Sigma Chemical Co. (St. Louis, MO); FCS from Gibco (Grand Island, NY);
deoxyribonuclease I and pronase from Calbiochem (La Jolla, CA); 60%
HClO4 from Baker Chemical Co. (Deventer, The
Netherlands); RPMI 1640 with 25 mmol/L Hepes buffer, Eagle's minimum
essential medium (MEM) from Flow Laboratories (Irvine, Scotland);
Dextran 70 and Percoll from Pharmacia Fine Chemicals (Uppsala, Sweden);
Alcian Blue 8GX from Carlo Erba (Milan, Italy); recombinant human stem
cell factor (SCF) from Amgen (Thousand Oaks, CA). PMA, ionomycin,
brefeldin A, anti-glycophorin A and B mAbs, and saponin were purchased
from Sigma. The PE-conjugated anti-IL-4 (3010.211, IgG1),
FITC-conjugated anti-IFN-
(25723.11, IgG2b), PerCP- and
PE-conjugated anti-CD4 as well as the purified anti-CD8, anti-CD14,
anti-CD20, anti-CD56 and anti-CD45Ro mAbs were purchased from Becton
Dickinson (San Jose, CA). The purified IgG1 and IgG2a isotype control
mAbs, as well as the FITC-conjugated anti-IgG2a and PE-conjugated IgG1
Abs, were purchased from Southern Biotechnology Associates (Birmingham,
AL). The goat anti-mouse IgG Abs conjugated with magnetic beads (MACS)
were purchased from Miltenyi Biotec (Bisley, Germany).
Buffers
The PIPES buffer used in the histamine release and chemotaxis experiments was made up of 24 mmol/L PIPES, pH 7.37, 110 mmol/L NaCl, 5 mmol/L KCl (P). The mixture which is referred to as P2CG contains in addition to P, human serum albumin (HSA) 3%, 2 mmol/L CaCl2, 1 g/l dextrose. pH was titrated to 7.4 with sodium bicarbonate. PACGM contains in addition to P, HSA 3%, 1 mmol/L CaCl2, 1 g/l dextrose and 0.25 g/l MgCl2x6H2O, pH 7.4; PGMD contains 0.25 g/l MgCl2x6H2O, 10 mg/l DNase, and 1 g/l gelatin in addition to P, pH 7.37.12,13
Immunohistochemical Analysis
Immunohistochemical analysis was performed on skin, gut, and lung tissues. Skin specimens were obtained from four patients with systemic sclerosis, one with lichen ruber planus and one with systemic mastocytosis, undergoing skin biopsy for diagnostic purpose. Gut specimens were obtained from biopsies of four patients with ulcerative colitis and from fragments of normal gut tissue of three patients undergoing colectomy because of colon cancer. Lung specimens were obtained from fragments of normal lung of three patients subjected to pneumectomy because of lung cancer. The procedures used in this study were in accordance with the criteria of the regional ethical committee on human experimentation.
Immunohistochemical staining was performed according to a technique previously described.14 To this end, 10-µm cryostat sections were fixed in 4% paraformaldehyde for 20 minutes and subsequently exposed to 0.3% hydrogen peroxide-methanol solution to quench endogenous peroxidase activity. After a 30-minute preincubation with normal horse serum (Vectastain ABC kit; Vector Laboratories, DBA, Milan, Italy), sections were layered for 30 minutes with anti-CCR3 (0.2 µg/ml), anti-CD3, (5 µg/ml), anti-PAM-1 (5 µg/ml), anti-ECP (2 µg/ml), anti-tryptase (0.4 µg/ml) or anti-chymase (0.5 µg/ml) Abs, followed by biotinylated anti-mouse IgG Ab, and the avidin-biotin-peroxidase complex (Vectastain ABC kit), as described.13 3-amino-9-ethylcarbazole (AEC; Vector) or Vector SG were used as peroxidase substrates. Finally, sections were counterstained with Gill's hematoxylin or Methyl Green and mounted with Kaiser's glycerol gelatin. All incubations were performed at room temperature. As negative control, primary mAb was replaced with an isotype-matched antibody with irrelevant specificity or mouse ascites fluid.
Double immunostaining was performed with anti-CCR3 and anti-CD3, anti-CCR3 and anti-ECP, anti-CCR3 and anti-tryptase, and anti-CCR3 and anti-chymase Ab by using the avidin-biotin-peroxidase system with two different substrates, as described.14 To identify the two molecules on the same specimen, the Vector SG (bluish-grey color) and the AEC (red color) substrates were used, respectively.
Generation of Th2-Oriented T Cell Lines from Umbilical Cord Blood (UCB) Lymphocytes
Polyclonal CD4+ T cell lines were generated from UCB mononuclear cell (MNC) suspensions of four donors, as described.15 Briefly, CD4+ CD45RA+ T cells were purified by negative magnetic selection using the MACS system after a two-step incubation with a mixture of anti-CD8, anti-CD14, anti-CD20, anti-CD56, antiCD45R0, anti-glycophorin A and B mAbs, followed by goat anti-mouse IgG conjugated with magnetic beads. Recovered cells (>99% CD3+ CD4+ CD45RA+) were then stimulated with PHA (0.1% vol/vol) and IL-2 (20 U/ml) in the presence of IL-4 (100 U/ml) in RPMI medium containing 10% heat-inactivated FCS (primary stimulation). On day 7, cells were washed and restimulated with PHA (0.1% vol/vol), IL-2 (20 U/ml), and IL-4 (100 U/ml) for 4 more days (secondary stimulation).
Detection of Intracellular IL-4 and IFN- Production by T Cell
Lines
Intracytofluorimetric analysis of IFN- and IL-4 synthesis at
the single-cell level was performed as described
elsewhere.15
Briefly, T cell blasts were stimulated with
PMA (10 ng/ml) plus ionomycin (1 µmol/L) for 4 hours, the last 2 of
which were in the presence of brefeldin A (5 µg/ml). After
incubation, cells were washed twice with PBS, pH 7.2, fixed 15 minutes
with formaldehyde (2% in PBS, pH 7.2), washed twice with PBS, pH 7.2,
permeabilized with PBS, pH 7.2, containing 0.5% BSA and 0.5% saponin,
then incubated with the appropriate mAb. Cells were analyzed on a
FACSCalibur cytofluorimeter using CellQuest software (Becton
Dickinson). The area of positivity was determined using an
isotype-matched Ab. A total of 104
events for each sample
were acquired in all cytofluorimetric analyses.
Purification of Peripheral Blood (PB) Basophils
Basophils were purified from PB cells of normal subjects undergoing hemapheresis. "Buffy coat" cell packs from healthy volunteers, provided by the Immunohematology Service at the University of Naples Federico II, were reconstituted in PBS containing 0.5 g/l HSA and 3.42 g/l sodium citrate, and loaded onto a countercurrent elutriator (model J2-21, Beckman Instruments, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 x 106 basophils) and of good purity (>15%) were further enriched by discontinuous Percoll gradients.16 Yields ranged from 3 to 10 x 106 basophils with purity usually higher than 65%, as assessed by basophil staining with Alcian Blue and counting in a Spiers-Levy eosinophil counter.16
Isolation and Purification of Lung MC
Lung tissue was obtained from 12 patients undergoing thoracotomy and lung resection. These samples were different from those used for immunohistochemical analysis. Macroscopically normal parenchyma was dissected free from pleura, bronchi, and blood vessels, and minced into a single-cell suspension, as previously described.12 Yields with this technique ranged between 3 x 106 and 18 x 106 MC, and purities were between 1% and 8%. Human lung MC were purified by countercurrent elutriation (J2-21, Beckman Instruments) and then by discontinuous Percoll density gradient, as previously described.12 The final preparations contained >95% viable cells, as assessed by trypan blue exclusion method, and consisted of 25 to 96% MC.
Flow Cytometric Analysis of Surface Molecules
Flow cytometric analysis of cell surface molecules was performed as described elsewhere.15 Briefly, after saturation of nonspecific binding sites with total rabbit IgG, cells were incubated for 20 minutes on ice with specific or isotype control Abs. For indirect staining this step was followed by a second incubation on ice with an appropriate anti-isotype-conjugated Ab. Finally, cells were washed and analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson). A total of 104 events for each sample were acquired in all cytofluorimetric analyses.
Histamine Release
Cells (~3 x 104
MC per tube) were
resuspended in P2CG, and 0.3 ml of the cell suspensions were placed in
12 x 75 mm polyethylene tubes (Sarstadt Inc., Princeton, NJ) and
warmed to 37°C; 0.2 ml of each prewarmed releasing stimulus was
added, and incubation was continued at 37°C.16
The P2CG
and PGMD buffers12,13
were used in these experiments. At
the end of this step, the reaction was stopped by centrifugation
(1,000 x g, 22°C, 1 minute), and the cell-free
supernatants were stored at -20°C for subsequent assay of histamine
content, with an automated fluorimetric technique.17
Total
histamine content was assessed by lysis induced by incubating the cells
with 2% HClO4 before centrifugation. To calculate
histamine release as a percentage of total cellular histamine, the
spontaneous release of histamine from mast cells (2% to 14% of the
total cellular histamine) was subtracted from both numerator and
denominator.13
The percentage of histamine release was
calculated according to the following equation:
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Chemotactic Assay
MC chemotaxis was performed using a modified Boyden chamber technique, as described previously.18 Briefly, 25 µl of PACGM buffer or various concentrations of the stimuli in the same buffer were placed in triplicate in the lower compartment of a 48-well micro-chemotaxis chamber (Neuroprobe, Cabin John, MD). The lower compartments were covered with a two-filter sandwich, a lower 5-µm pore size and an upper 8-µm pore size polycarbonate membranes (Nucleopore Corp., Pleasanton, CA), and then 50 µl of the cell suspension (5 x 104/well) resuspended in PACGM were pipetted into the upper compartments. The chemotactic chamber was then incubated for 3 hours at 37°C in a humified incubator with 5% CO2 (Automatic CO2 Incubator, Model 160 IR, ICN Flow). After the incubation period, the upper polycarbonate filter was discarded, while the lower nitrate cellulose filters were fixed in methanol, stained with Alcian Blue,19 and then mounted on a microscope slide with Cytoseal (Stephen Scientific, Springfield, NJ). MC chemotaxis was quantitated microscopically by counting the number of cells that had traversed the upper 8-µm polycarbonate filter and were attached to the surface of the 5-µm cellulose nitrate filter. In each experiment, ten fields per triplicate filter were measured at 40x magnification. The results were compared with buffer controls.
To discriminate between chemotaxis and nondirected migration (chemokinesis) of MC, checkerboard analyses were performed. In the experiments, MC were placed in the upper chambers and various concentrations of SCF (10 to 100 ng/ml), eotaxin (10 to 100 ng/ml), RANTES (10 to 100 ng/ml) or PACGM buffer was added into either the upper or lower wells or into both. Spontaneous migration (chemochinesis) was determined in the absence of chemoattractant or when stimuli were added into either the lower and upper chambers. The MC migratory response to SCF, RANTES, and eotaxin was largely due to chemotaxis and not to chemokinesis. Indeed, a checkerboard analysis, in which chemoattractants above and below the filter were varied, resulted in significant migration only when there was a gradient of the factor below the filters (data not shown).
Statistical Analysis
The results are expressed as means ± SE. Statistical significance was analyzed by one-way analysis of variance and, when the F value was significant, by Duncan's multiple range test. Differences were considered significant when P < 0.05.
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Results |
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We used immunohistochemistry to investigate CCR3 expression in
human skin, gut, and lung tissues. Several CCR3+ cells were detected in
all tissues, but were more abundant in the skin and in the intestinal
submucosa than in intestinal mucosa or in the lung (Figure 1 A–D)
. To identify the nature of CCR3+
cells in the various tissues, we used double immunostaining with
anti-CCR3 and anti-CD3 Ab (for T cells) or CCR3 and anti-ECP Ab (for
eosinophils). Cells staining for both CCR3 and ECP were absent from
normal tissues, but they were clearly detectable in the gut of patients
with ulcerative colitis. However, several CCR3+ cells in the gut of
these patients, as well as the vast majority of those present in the
other tissues tested, were ECP-negative (Figure 1E)
. Cells staining for
both CCR3 and CD3 were virtually absent from every tissue examined,
including the gut of patients with ulcerative colitis (data not shown),
where CCR3+ T cells have been reported,9
and the skin of
patients with systemic sclerosis (Figure 1F)
, where the majority of
infiltrating T cells express a Th2-type cytokine profile.20
Morphologically, the CCR3+ ECP- CD3- cells present in all tissues
examined were usually larger than lymphocytes and eosinophils. In an
attempt to identify these cells, we double-stained them with CCR3 and
PAM-1, which is expressed by macrophages and dendritic
cells.11
As shown in Figure 1G
, CCR3 and PAM-1 staining was
clearly distinct, indicating that these cells do not belong to the
macrophage lineage.
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. Therefore, we performed
double immunostaining for tryptase and CCR3 on normal lung, skin, and
gut specimens. In all tissues examined, variable proportions of
tryptase-positive cells showed costaining for CCR3. A representative
experiment showing double immunostaining for tryptase and CCR3 in the
submucosa of normal gut is shown in Figures 2, C and D
.
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RI+ cells in basophil-enriched PB suspensions
(Figure 3A)
and in a remarkable
proportion of FcRl+ cells (25%) present in MC-enriched lung
suspensions (Figure 3B)
. By contrast, despite their clearly polarized
Th2 profile of cytokine production, short-term T cell lines generated
from UCB T lymphocytes did not show perceptible reactivity with the
same anti-CCR3 mAb (Figure 3C)
.
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To establish whether CCR3 expressed by a percentage of MC was
functionally active, MC-enriched suspensions obtained from fragments of
lung tissues were tested for their ability to release histamine in
response to eotaxin, which selectively binds to CCR3. The effect of
eotaxin on histamine release from MC-enriched suspensions was compared
with the effect induced by an anti-IgE Ab, the most potent
immunological stimulus of human lung MC mediator release. Eotaxin alone
elicited poor or no response and did not enhance the histamine release
induced by anti-IgE Ab (Figure 4)
.
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, to act as
chemoattractants for MC-enriched preparations with that of SCF. Eotaxin
induced strong attraction of MC and its chemoattractant activity, as
expressed by the number of migrating MC at optimal concentration, was
similar to that of RANTES and SCF (Figure 5)
. By contrast, MIP-1 was ineffective
(data not shown).
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shows that
pre-incubation of MC with anti-CCR3 Ab completely blocked the
chemoattractant effects of both eotaxin and RANTES.
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Because the percentage of CCR3-expressing cells in normal tissues
was usually lower than that of tryptase-positive cells, we investigated
whether CCR3 was expressed by a subset of MC, and examined the nature
of this MC subset. To this end, the percentages of tryptase-positive
cells showing CCR3 expression in biopsy specimens from gut, lung, and
skin were evaluated. High percentages (>70%) of tryptase-positive
cells showing CCR3 expression were found in the skin and in the
intestinal submucosa, whereas much lower percentages (
20%) were
found in the intestinal mucosa and in lung interstitium (Table 1)
. This suggests that CCR3 is
preferentially expressed by MC present in connective tissue rather than
in mucosal MC. Because tryptase is present in the vast majority of
human MC, whereas chymase is localized to MC predominantly associated
with connective tissues,21,22
we asked whether CCR3
expression by human MC was a selective property of MCTC. To
test directly this possibility, we double stained gut, lung, and skin
specimens with anti-CCR3 and anti-chymase Ab. The vast majority of
CCR3+ MC in the tissues examined also stained positive for chymase, the
proportion of those reactive with chymase, but not with CCR3, being
lower than 1%. A representative experiment, showing double
immunostaining for chymase and CCR3 in the intestinal submucosa, is
depicted in Figure 7
.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our finding that polyclonally activated Th2-polarized naive T cells, as well as Th2 cells present in the skin of patients with systemic sclerosis,20 do not express CCR3 is apparently at variance with reports of CCR3 expression by human Th2 cells in vitro and in vivo.8,9 The reason for this discrepancy is presently unclear, since in our experiments the same anti-CCR3 Ab (7B11) already reported to be reactive with Th2 cells8,9 was used. However, the present finding is consistent with recent data showing poor or no CCR3 expression by human Th2-polarized T lymphocytes,23-25 as well as with the demonstration that there are virtually no CCR3-expressing T cells in bronchial biopsy specimens of patients with atopic asthma,26 where Th2 cells have been shown to play a pathogenic role.27 Thus, it can be concluded that CCR3 expression by Th2 cells both in vitro and in vivo is too faint to be revealed by either flow cytometry or immunocytochemistry.
The results of our study suggest, rather, that the other cell type
expressing CCR3 in human tissues in addition to eosinophils (and
possibly basophils) is not a Th2 cell, but a MC. First, the assessment
of skin biopsy specimens from one patient with systemic mastocytosis,
showing diffuse MC dermal infiltration, revealed CCR3 expression in a
remarkable proportion of these cells. More importantly, double
immunostaining for CCR3 and tryptase in skin, gut, and lung, as well as
double labeling for FcRI and CCR3 on MC-enriched suspensions
obtained from normal lung, confirmed the MC nature of the majority of
CCR3-expressing cells in different tissues.
MC are not a homogeneous population, but show marked inter- and intraspecies differences.28-31 In rodents, metachromatic staining differentiated mucosal MC (MMC) from connective tissue MC (CTMC).30,31 Although a similar subdivision has been suggested in humans, the histochemical subtype appears to be unrelated with anatomical site because both subtypes are found in the skin32,33 and in the gut.34-36 An immunohistochemical subtyping of human MC has been proposed based on the presence of neutral proteases (tryptase and chymase) in these cells. Tryptase is present, in different concentrations, in the vast majority of human MC from different tissues,21,22 whereas it was reported that chymase is present only in MC associated with connective tissue.33 Thus, an immunocytochemical subtyping of MC into MCT (MC containing only tryptase) and MCTC (MC containing both tryptase and chymase) has .21,37 MCT are preferentially located at mucosal surfaces, whereas MCTC are found predominantly in submucosal and connective tissues.38 The results of our study suggest that CCR3 expression is mainly a property of MCTC. First, CCR3+ MC were more abundant in skin derma and intestinal submucosa than in intestinal mucosa and lung interstitium and their proportions in these tissues roughly approximated those of MCTC. More importantly, double immunostaining for CCR3 and chymase showed that the vast majority of chymase-positive MC also stained for CCR3.
In this study, chemotaxis assay yielded essential information about the
functional role of CCR3 in MC. First, eotaxin (a selective ligand for
CCR3)1-3,39
and RANTES (which also binds to CCR1, CCR5 and
CCR9)1-3,39
acted as chemoattractants for human MC as SCF,
whereas MIP-1 (which binds to CCR1, CCR5, and CCR9, but not to
CCR3)1-3,39
was inactive. This suggests that MC, like
human basophils and eosinophils, express functional CCR3. More
importantly, in vitro, anti-CCR3 Ab abrogated chemotaxis
elicited by eotaxin. Therefore, eotaxin appears to be a selective
agonist for CCR3 on human MC, as well as on eosinophils4-6
and basophils.7
The inhibitory effect of the anti-CCR3
antibody indicates that CCR3 is the predominant receptor for MC
migration in response not only to eotaxin, but also to RANTES. In
contrast to the effects exerted on chemotaxis, histamine release was
poorly or not at all sensitive to eotaxin, suggesting that CCR3 may
primarily mediate migration rather than mediator secretion by human MC.
These results are in agreement with the observation that eotaxin is a
much better stimulus of human basophil chemotaxis than of histamine and
leukotriene release.7
Mediator release was induced by
eotaxin only in IL-3-primed basophils and at concentrations 10 to 100
times greater than those used here. Thus, from this study CCR3 emerges
as a major receptor for the eotaxin- and RANTES-mediated recruitment
not only of eosinophils and basophils, but also of human MC.
Interestingly, in vivo injection of human
recombinant RANTES in mice has been shown to cause MC
recruitment.40
Why CCR3 is primarily expressed by MCTC, which predominate in connective tissues rather than in the mucosa, is unknown, also because the mechanisms that regulate the in situ differentiation of human MC are largely unclear. It has been proposed that MC subtypes represent different stages of differentiation of a single cell line,41 whereas others suggest that they derive from distinct precursors.38 In rodents, Kitamura and coworkers demonstrated that the development of various phenotypes depends on the anatomical microenvironment where the final differentiation takes place.42,43 It is noteworthy that in mice both eotaxin and CCR3 are expressed by embryonic tissues responsible for blood development, eg, fetal liver, yolk sac, and peripheral blood.44 Moreover, in combination with SCF, eotaxin promotes the growth and differentiation of MC progenitors.44 Finally, eotaxin increased significantly during fibroblast-MC interaction, which appeared to be dependent upon SCF production.45
Some years ago, Furitsu et al showed that prolonged coculture of human UCB nucleated cells with skin fibroblasts results in the development of mature MC, mostly MCTC.46 This suggests that fibroblasts not only facilitate the differentiation of MC precursors to mature MC, but also contribute to the determination of the MCTC phenotype.46 Interestingly, murine MC, which develop in response to the combined action of SCF and eotaxin, also exhibit the phenotype of connective-tissue-type MC.47 Because eotaxin is constitutively expressed even in nonimmune tissues, such as intestine, skin, and mammary gland,48 where the vast majority of MC are MCTC,29 it is tempting to speculate that this chemokine not only plays an important role in the preferential differentiation of MC precursors into the MCTC phenotype, but also favors their migration into the connective tissues and helps maintaining their differentiation pathway and/or their survival. Furthermore, the demonstration that, like basophils and eosinophils, a proportion of human MC express CCR3 and therefore can respond to eotaxin, as well as other agonistic cytokines, may account for their up-regulation in tissues known to be sites of allergic reactions such as the airways. Indeed, in vivo administration of RANTES induced MC hyperplasia40 and high eotaxin expression occurs in the epithelium and submucosa of bronchial biopsies from patients with atopic asthma.26 Thus, the finding reported here may open new avenues for the identification of mechanisms involved in MCTC differentiation and homing, and possibly for a better understanding of their pathophysiological significance.
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Acknowledgements |
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Footnotes |
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Supported by grants provided by Istituto Superiore Di Sanita (AIDS Project to S. R. and G. M.) and by C. N. R. (Target Project Biotechnology Nos. 97.01140.PF49 and 98.00085.PF31) to G. M.
Accepted for publication June 30, 1999.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis. J Clin Invest 1995, 96:2702-2710
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L. Pang, M. Nie, L. Corbett, A. Sutcliffe, and A. J. Knox Mast Cell beta-Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities J. Immunol., March 15, 2006; 176(6): 3788 - 3795. [Abstract] [Full Text] [PDF]
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G. F. Debes, M. E. Dahl, A. J. Mahiny, K. Bonhagen, D. J. Campbell, K. Siegmund, K. J. Erb, D. B. Lewis, T. Kamradt, and A. Hamann Chemotactic Responses of IL-4-, IL-10-, and IFN-{gamma}-Producing CD4+ T Cells Depend on Tissue Origin and Microbial Stimulus J. Immunol., January 1, 2006; 176(1): 557 - 566. [Abstract] [Full Text] [PDF]
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A. de Paulis, N. Montuori, N. Prevete, I. Fiorentino, F. W. Rossi, V. Visconte, G. Rossi, G. Marone, and P. Ragno Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2 J. Immunol., November 1, 2004; 173(9): 5739 - 5748. [Abstract] [Full Text] [PDF]
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A. de Paulis, N. Prevete, I. Fiorentino, A. F. Walls, M. Curto, A. Petraroli, V. Castaldo, P. Ceppa, R. Fiocca, and G. Marone Basophils Infiltrate Human Gastric Mucosa at Sites of Helicobacter pylori Infection, and Exhibit Chemotaxis in Response to H. pylori-derived Peptide Hp(2-20) J. Immunol., June 15, 2004; 172(12): 7734 - 7743. [Abstract] [Full Text] |
), eotaxin
(
), and RANTES
(
) on MC chemotaxis.
MC-enriched suspensions obtained from human lung were allowed to
migrate toward the indicated concentrations of SCF, eotaxin, and RANTES
for 3 hours at 37°C in a humidified incubator with 5%
CO2. Values are the mean
(±SE) of four distinct
experiments with different human lung MC-enriched preparations.
P < 0.01 when compared with control.
