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Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London, United Kingdom; and
LeukoSite, Inc., Cambridge, MA 02142
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1), and IL-8 on leukocytes in mixed cell suspensions and
focused on the responses of eosinophils to C-C chemokines. Those
chemokines acting on CCR3 induced a rapid shape change in eosinophils
from all donors; of these, eotaxin and eotaxin-2 were the most potent.
Responses to MCP-4 were qualitatively different, showing marked
reversal of shape change responses with agonist concentration and
duration of treatment. In contrast, MIP-1 induced a potent response
in eosinophils from a small and previously undescribed subgroup of
donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of
leukocytes at 37°C for 90 min in the absence of extracellular calcium
up-regulated responses to MCP-4 and MIP-1 in the majority of donors,
and there was a small increase in responses to eotaxin. MIP-1
responsiveness in vivo may therefore be a function of both CCR1
expression levels and the regulated efficiency of coupling to
intracellular signaling pathways. The observed up-regulation of
MIP-1 signaling via non-CCR3 pathways may play a role in eosinophil
recruitment in inflammatory states such as occurs in the asthmatic
lung. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using primers based on the guinea pig sequence, human eotaxin has been cloned and its synthesis (mRNA and protein) demonstrated in allergic diseases including asthma 7, 8, 9, 10 . Recently, another human eosinophil-selective C-C chemokine has been described and named eotaxin-2 on the basis of its functional homology with eotaxin 11, 12 . Eotaxin and eotaxin-2 are potent stimulators of eosinophils, signaling exclusively via high affinity binding to the receptor CCR34, 13 . C-C chemokines, including MCP-3, MCP-4, and RANTES, also stimulate eosinophils via CCR3, although they show less leukocyte selectivity as they additionally signal via other chemokine receptors expressed on a range of leukocytes, including lymphocytes and monocytes 14, 15, 16, 17 . CCR3 is expressed in high numbers on eosinophils 13, 18 and is thought to be the major eosinophil chemokine receptor. Blockade of CCR3 in vivo inhibits eosinophil recruitment in response to eotaxin in both the guinea pig 19 and mouse 20 . More recently, CCR3 expression has been demonstrated on basophils 16 and Th2-type T cells 21, 22, 23, 24 , suggesting possible roles for CCR3 in the genesis and maintenance of allergic inflammation. CCR3 is therefore a major target for anti-inflammatory drug development 25 .
There is evidence that eosinophils express low levels of CCR1 13, 18 ,
a receptor for MIP-1, RANTES, and MCP-3. MIP-1 was first isolated
from LPS-stimulated macrophages 26 and is thought to be a
preferential monocyte chemoattractant 27 , acting via CCR1 28 or
CCR5 29 . Although MIP-1 is among the chemokines produced in the
human asthmatic lung in response to allergen 30, 31 , there is
controversy over its ability to induce functional responses in
eosinophils 32, 33, 34 .
Leukocyte chemoattractants such as eotaxin initiate a coordinated sequence of adhesive interactions between cells in the circulation and the microvascular endothelium. The phases of leukocyte migration comprising leukocyte adhesion, spreading, penetration of the vessel wall, and tissue infiltration all depend on a continual reorganization of cytoskeletal elements associated with specific changes in cell morphology 35, 36, 37 . The formation of microvillae, uropodia, and lammelipodia is important in regulating the distribution and accessibility of cell surface adhesion molecules that are involved in cell migration 37, 38 .
We postulated that analysis of the leukocyte shape change response to
chemoattractant agonists would be a powerful tool for analyzing
receptor usage by chemokines in human leukocytes. We have developed a
sensitive flow cytometric system to quantitate shape changes in
eosinophils and neutrophils simultaneously in mixed cell suspensions,
the gated autofluorescence/forward scatter (GAFS) assay. This system
uses differential autofluorescence to identify leukocyte type and
changes in forward scatter (FSC) to measure shape change in response to
agonists. We have characterized the shape change responses of
eosinophils, neutrophils, and monocytes in response to a range of
chemokines. Using the GAFS assay we have discovered considerable
variations in the responses of eosinophils from individual donors to
MIP-1 and demonstrated that these responses may be up-regulated by a
simple preincubation of the leukocytes in the absence of calcium. A
group of donors was also identified whose eosinophils consistently
responded to MIP-1 with a potency comparable to that of eotaxin. The
existence of these signaling pathways is of significance in the
development of small molecule chemokine receptor antagonists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BSA, EDTA, and glucose were purchased from Sigma (Poole, U.K.).
RPMI 1640, FCS, PBS, and HEPES were purchased from Life Technologies
(Paisley, U.K.). Dextran T-500 and Percoll were purchased from
Pharmacia (St. Albans, U.K.). FITC-labeled goat anti-mouse
polyclonal F(ab')2 were obtained from Dako (High Wycombe,
U.K.). IgG1 (clone MOPC 21) and IgG2a (clone UPC 10) negative control
Abs were obtained from Sigma. Human recombinant RANTES, MIP-1,
MCP-1, MCP-3, and MCP-4 were purchased from PeproTech (London, U.K.).
Anti-CD16 microbeads were purchased from Miltenyi-Biotech (Bergisch
Gladbach, Germany). Cellfix was obtained from Becton Dickinson
(Mountain View, CA). Anti-human CCR3 mAb 7B11 13, 34 , anti-human
CCR1 mAb 2D4, and recombinant human eotaxin were produced by LeukoSite
(Boston, MA). The following were generous gifts: recombinant human IL-8
and eotaxin-2 were from Dr. J. White, SmithKline Beecham (King of
Prussia, PA); recombinant human C5a was from Dr. J. van Oostrum
(Ciba-Geigy, Summit, NJ); anti-human IL-8RA (CXCR1) and IL-8RB
(CXCR2) mAbs were from Dr. C. A. Hébert (Genentech, South
San Francisco, CA); and anti-VLA-4 mAb was from Dr. R. R. Lobb
(Biogen, Cambridge, MA).
Cell preparation
Volunteer blood donors were healthy normal subjects or atopics, as defined by a history of asthma, eczema, or hayfever, and symptoms on exposure to common aero-allergens, including pollens and house dust mites. The donors were taking no systemic medication. Blood was sampled according to a Royal Brompton Hospital ethical committee-approved protocol and was prepared as previously described 39 . Briefly, platelet-rich plasma was removed by centrifugation of citrated whole blood, after which the erythrocytes were removed by dextran sedimentation. Polymorphonuclear leukocytes (PMNL; containing neutrophils and eosinophils) were then separated from PBMC over a discontinuous plasma-Percoll gradient. Any erythrocyte contamination of the PMNL pellet was removed by hypotonic shock lysis 40 . After preparation, an aliquot of cells was stained with Kimura stain and counted. Eosinophil counts ranged from 2–15% of the total PMNL number. In some experiments (as indicated in Results) eosinophils were further purified from the PMNL pellet by negative magnetic selection, where the leukocytes were incubated with anti-CD16-coated microbeads in RPMI, 0.5% BSA, 20 mM HEPES, and 5 mM EDTA, which selectively bound neutrophils in the PMNL suspension. The eosinophils were separated from the neutrophils by passage of the cell suspension through a magnetic field 41 , resulting in eosinophil populations of >97.5% purity; the contaminating cells were PBMCs.
Measurement of changes in leukocyte shape using flow cytometry
For the standard GAFS assay, purified PMNL were washed in buffer (10 mM PBS containing Ca2+ and Mg2+, 10 mM HEPES, 10 mM glucose, and 0.1% BSA, pH 7.2–7.4) and preincubated for 30 min at 37°C. In additional experiments as indicated in Results, the cells were preincubated for 30 or 90 min in the presence or the absence of Ca2+ and Mg2+. In all experiments after preincubation the cells were washed again in buffer containing Ca2+ and Mg2+, resuspended in buffer containing Ca2+ and Mg2+, and held for 5 min at room temperature to allow equilibration of intracellular and extracellular calcium. Aliquots of cells (5 x 105 PMNL) were mixed with agonists or buffer in 1.2-ml polypropylene cluster tubes (Costar, Cambridge, MA) in a final volume of 100 µl. The tubes were placed in a 37°C shaking water bath for 6 min (or as indicated), after which they were transferred to an ice-water bath, and 250 µl of ice-cold optimized fixative (a 1/4 dilution of 1x Cellfix-buffered formaldehyde solution in PBS) was added to terminate the reaction and maintain the cell shape change until analysis. Initial experiments demonstrated that basal cell FSC and agonist-induced changes in cell FSC were not affected by fixation using this regimen, but were maintained without alteration for up to 2 h poststimulation on ice. In the absence of fixation, changes in cell FSC were identical with those seen in the fixed cell populations, but persisted for only 40 min when the cells were held on ice. In some experiments the cell aliquots or agonists (5 x 105 PMNL) were incubated for 10 min with Abs or buffer in 50 µl before addition of the other components of the assay (also in 50 µl to a final volume of 100 µl as above). The samples were then analyzed immediately on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) whose FSC detector had been optimized using control 2-µm beads and resting and stimulated leukocytes. Data was acquired using the FL-2 fluorescence channel on a sensitive setting, allowing eosinophils to be distinguished from neutrophils by their higher autofluorescence. FSC, side scatter (SSC), and FL-2 data were acquired, and acquisition was terminated after 500 high fluorescence (eosinophil) events.
In experiments investigating the effects of cation chelation during the preincubation, PMNL were preincubated with buffer (containing Ca2+ and Mg2+) and either 5 mM EDTA or 2 mM EGTA for 90 min in parallel with PMNL preincubated in buffer both with and without Ca2+ and Mg2+. After the preincubation, all cells were washed twice in buffer to remove the chelating agents. All cells were finally washed once in buffer containing Ca2+ and Mg2+ before performance of the GAFS assay as described above. Buffer pH was checked before and after the preincubation and did not vary over the course of the experiment (data not shown).
All data presented here show the responses of PMNL and PBMC in the presence of Ca2+ and Mg2+; manipulation of responses by removal of cations occurred during the preincubation phases only.
To measure monocyte shape change in response to chemokines, a similar protocol was used. However, since monocytes could not be identified separately from other cells by autofluorescence, an additional labeling step was incorporated. PBMCs were incubated for 10 min at room temperature with FITC-labeled anti-CD14 mAb (PharMingen, San Diego, CA) at 1 x 107/ml, and washed once. After labeling, determination of monocyte shape change proceeded as described for PMNL above, except that agonist stimulation was for 10 min at 37°C, and data were acquired using the FITC FL-1 channel to identify monocytes. One thousand monocyte events were counted for each sample.
Immunofluorescence flow cytometry
To confirm eosinophil and neutrophil identity as predicted by autofluorescence, mixed PMNL were suspended in buffer at 5 x 106 cells/ml. Samples were incubated on ice for 60 min with saturating concentrations of anti-CXCR1 mAb, anti-CXCR2 mAb, and anti-VLA-4 mAb; washed; and then incubated with FITC-conjugated polyclonal goat anti-mouse F(ab')2 Abs on ice for 30 min. Nonbinding control mAbs of IgG1 and IgG2a subclasses were used to determine nonspecific binding. Samples were held on ice until FACS analysis. For staining of eosinophils in whole blood, aliquots of EDTA-anticoagulated blood (100 µl) were incubated for 1 h on ice with 10 µg/ml anti-CCR1 mAb 2D4, 3 µg/ml anti-CCR3 mAb 7B11, or relevant isotype-matched control Abs. The blood was then washed with buffer (PBS and 0.25% BSA) and resuspended in 100 µl of FITC-conjugated polyclonal goat anti-mouse F(ab')2 Abs on ice for 30 min. After one additional wash the red cells were lysed using a commercial lysis preparation according to the manufacturer’s instructions (Optilyse B, Coulter, Luton, U.K.), and the samples were held on ice until FACS analysis.
Measurement of cytosolic calcium
Eosinophils purified by immunomagnetic selection (1 x 107/ml) were loaded with 1 µM fura-2 in PBS without Ca2+ for 30 min at 37°C, washed in PBS and 0.1% BSA, and resuspended at a final concentration of 1 x 106 cells/ml in PBS, 0.1% BSA, 10 mM HEPES, and 10 mM glucose. Aliquots of cells (2 ml) were dispensed into quartz cuvettes and equilibrated with 1 mM calcium at 37°C for 12 min before use. Changes in fluorescence were measured in a Perkin-Elmer LS-50 fluorescence spectrophotometer (Norwalk, CT) as previously described 40 . Calculation of intracellular free calcium was derived from the fluorescence spectra (excitation wavelengths, 340 and 380 nm; emission wavelength, 510 nm) in accordance with established methods 40 .
Statistical analysis
Data were analyzed using analysis of variance and Bonferroni’s posttest.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
A shows a dot plot
of SSC vs FSC and the FL-2 fluorescence histogram of unstained,
unstimulated PMNL (eosinophils and neutrophils). The cells were divided
into two distinct populations of high and low autofluorescence (R1 and
R2, respectively) on the fluorescence histogram, correlating with the
proportion of eosinophils and neutrophils, respectively, as determined
in Kimura-stained preparations (data not shown). The light scatter
characteristics of the eosinophils and neutrophils within the
population (as identified by autofluorescence) are shown separately,
and from these plots the mean FSC of each population is determined. We
confirmed the identity of these leukocyte populations using Abs to
specific cell surface molecules. Fig. 1B shows the
fluorescence histograms of PMNL populations stained with
anti-VLA-4, anti-CXCR1, or anti-CXCR2 mAbs, adjacent to
which the light scatter characteristics of the stained cell populations
are shown. The predominant VLA-4-positive cell population (R3) showed
light scatter characteristics indistinguishable from those of the high
autofluorescence cells (R1; Fig. 1A), confirming that these
were eosinophils. Similarly, a single population of cells (R4) was
stained with anti-CXCR1 Ab, which showed the same light scatter
characteristics as the low autofluorescence cells, confirming their
identity as neutrophils (R2; Fig. 1A). Anti-CXCR2 labeled
two populations of cells. The most intense fluorescence was associated
with neutrophils (data not shown), while eosinophils also stained
weakly with this Ab (R5).
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Ci, shows the control unstimulated population for
comparison (eosinophil mean FSC = 208; neutrophil mean
FSC = 214; as in Fig. 1A). Fig. 1Cii, shows
that after stimulation with 0.3 nM eotaxin there was a selective
increase in eosinophil mean FSC (348), but not neutrophil mean FSC
(190) compared with those in the unstimulated cells. Conversely, there
was an increase in neutrophil mean FSC (331) but not in eosinophil mean
FSC (186) following addition of 0.3 nM IL-8 (Fig. 1Ciii),
while Fig. 1Civ, shows that C5a (1 nM) increased both
eosinophil and neutrophil mean FSC (252 and 373, respectively). There
were no significant changes in the mean SSC following any treatment,
and leukocyte autofluorescence was unaffected by chemokine stimulation.
Eosinophil shape change was not inhibited when the agonist stimulation
was performed in the absence of Ca2+ and Mg2+
(data not shown). In other experiments performed simultaneously with the investigation of PMNL responses to chemokines, PBMCs from the same donors were labeled with an FITC-conjugated anti-CD14 mAb to distinguish monocytes from lymphocytes before measurement of their FSC as described for PMNL above.
Eosinophil, neutrophil, and monocyte shape change induced by chemokines
In parallel experiments, the responses of eosinophils,
neutrophils, and monocytes to chemokines were determined using the
standard GAFS assay described above. Fig. 2A shows the percent change in
mean FSC of eosinophils from a mixed PMNL preparation in response to
the C-C chemokines eotaxin, eotaxin-2, MCP-1, MCP-3, and MCP-4 compared
with that in response to the C-X-C chemokine IL-8. Eotaxin, eotaxin-2,
and MCP-4 were the most potent inducers of eosinophil shape change,
followed by MCP-3, while MCP-1 and IL-8 were inactive. MCP-4 alone
showed a bell-shaped eosinophil dose-response curve over the dose range
tested. Eotaxin and eotaxin-2 were inactive on monocytes (Fig. 2B), whereas MCP-1, -3, and -4 all induced monocyte shape
change, with MCP-3 exhibiting the highest potency. Of the chemokines
tested, only IL-8 induced a change in neutrophil FSC (Fig. 2C). Eosinophil shape change in response to eotaxin was
abolished by pretreatment of the cells for 10 min at room temperature
with 10 µg/ml of anti-CCR3 mAb, 7B11 (see Fig. 6). Neutrophil and
eosinophil shape change responses to all agonists were abolished by
pretreatment of the cells for 30 min with cytochalasin B at 5 µg/ml
(data not shown).
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To investigate the time course of agonist-induced changes in cell
shape, PMNL and anti-CD14-labeled PBMC were incubated at 37°C
with eotaxin (2.5 nM), eotaxin-2 (2.5 nM), MCP-1 (10 nM), or MCP-4 (2.5
nM for PMNL, 10 nM for PBMC) for varying time periods from 0–20 min.
Fig. 3 shows that changes in eosinophil
and monocyte FSC occurred rapidly and could be detected by flow
cytometry within 30 s. For eosinophils stimulated by eotaxin or
eotaxin-2, maximal shape change was obtained after 4 min of chemokine
treatment and remained constant thereafter. The increase in eosinophil
FSC induced by MCP-4 gradually reversed on prolonged stimulation. This
reversal of shape change with time was observed with MCP-4 only. Both
MCP-1 and MCP-4 induced a rapid-onset, persistent, monocyte shape
change, with no reversal of stimulated cells to baseline shape up to
the 20 min point.
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Investigation of atopic individuals using the GAFS assay
(incorporating the standard 30-min preincubation step in the presence
of Ca2+ and Mg2+) identified a group whose
eosinophils showed strong MIP-1 responses (designated
MIP-1-highly responsive (MHR) individuals). These responses were in
marked contrast to the majority of individuals who showed absent or
poor MIP-1 responses under standard conditions (designated
MIP-1-poorly responsive (MPR) individuals). Eosinophils from the MHR
individuals showed a shape change in response to MIP-1 of similar
potency to that of eotaxin (Fig. 4).
There was no difference in responses to eotaxin or MCP-4 between MPR
and MHR individuals (Figs. 4, A and B, and
5). Sixteen individuals were investigated
for the ability of their eosinophils to respond to MIP-1, of whom 13
(81%) were MPR and 3 (19%) were MHR individuals. In contrast, there
was no significant difference in MIP-1 responsiveness in the
monocyte populations from either group screened on the same day as the
PMNL populations (Fig. 4, C and D).
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in the MHR group of
individuals, PMNL populations from each group were pretreated with the
blocking anti-CCR3 mAb 7B11 or a control mAb (MOPC 21) following
the 30 min preincubation step. 7B11 treatment completely blocked the
eosinophil responses to eotaxin (see Fig. 6) and MCP-4 (data not shown) from both
the MPR and MHR individuals. In contrast, MHR eosinophils showed a
potent response to MIP-1 that was not blocked by anti-CCR3 mAb.
The shape change response to RANTES was completely inhibited by
anti-CCR3 mAb pretreatment in the MPR individuals; however, in the
MHR group there was only partial inhibition of the response to RANTES
by CCR3 blockade (Fig. 6, A and B). Flow
cytometric analysis of eosinophil CCR1 and CCR3 expression was
performed in whole blood from four MPR donors and one MHR donor. These
data showed low levels of CCR1 expression on eosinophils from all
donors, in contrast to the expression of CCR3, which was at high levels
that were remarkably consistent between donors. Levels of eosinophil
CCR1 expression appeared higher in the MHR donor than in the MPR donors
(Fig. 7).
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Intracellular calcium flux in response to MIP-1 and eotaxin was
assessed in purified eosinophils prepared by negative magnetic
selection from one MPR and one MHR individual. Shape change was
simultaneously measured in the purified eosinophils to confirm the
MIP-1 responsiveness of the donors (data not shown). Fig. 8 shows that eosinophils from both MPR
and MHR individuals responded with similar calcium flux to 3 nM eotaxin
(Fig. 8, A and B). Eosinophils from the MPR
individual showed only a minimal calcium response to 10 nM MIP-1
(Fig. 8C), whereas in contrast, eosinophils from the MHR
individual showed a pronounced calcium flux in response to 10 nM
MIP-1 (Fig. 8D). Pretreatment with the anti-CCR3 mAb
7B11 (10 µg/ml) blocked eotaxin signaling in both individuals, but
not MIP-1 signaling in the MHR eosinophils (Fig. 8, E and
F). MOPC-21 pretreatment did not affect intracellular
calcium signaling in either group in response to these ligands (Fig. 8, C and D). Eosinophils from both the MPR and MHR
individuals showed similar intracellular calcium signaling responses to
C5a, and two additional MPR individuals showed similar overall patterns
of C-C chemokine responsiveness in assays of intracellular calcium flux
(data not shown).
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Pilot data suggested that eosinophil responses to chemokines
might be affected by variations in the preincubation conditions. Fig. 9 shows the eosinophil responses to
eotaxin, MCP-4, and MIP-1 after variations in these conditions.
Mixed PMNL were preincubated according to the standard GAFS assay
protocol (30 min in the presence of Ca2+ and
Mg2+). In parallel, PMNL from the same preparations were
also preincubated for 30 min in the absence of cations and for 90 min
in both the presence and the absence of cations. Subsequently, all GAFS
assays were performed as described previously, the only variations
between cell treatments being in the preincubation steps, and data were
acquired immediately after stimulation of the cells with agonists. Fig. 9 shows that responses to eotaxin, MCP-4, and MIP-1 were affected by
the preincubation conditions. Fig. 9B shows that responses
to 1.25 nM MCP-4 were greatest in the cells preincubated for 90 min
without Ca2+ and Mg2+
(p < 0.001). The order of responsiveness of
the eosinophils to MCP-4 for the different preincubation conditions was
90 min without cations > 30 min without cations > 30 min
with cations > 90 min with cations. Fig. 9C shows that
eosinophil responses to MIP-1 were absent or poor in this donor
group after the 30-min incubation with Ca2+ and
Mg2+. These responses to MIP-1 were significantly
up-regulated after preincubation for 90 min without cations
(p < 0.01 at the 0.6- and 1.25-nM
concentrations), although with greater variability between donors than
was seen with MCP-4 or eotaxin. An enhancement of eosinophil responses
to MIP-1 of varying magnitude occurred in seven of eight donors
tested, with one donor only showing no increase in MIP-1
responsiveness after the extended preincubation step. Eosinophil
responses to MIP-1 were similarly enhanced when either EDTA (5 mM)
or EGTA (2 mM) were added to the cells during the 90-min preincubation
with Ca2+ and Mg2+ (n = 4).
Shape change responses to both eotaxin and MIP-1 were abolished by
pretreatment of the PMNL with pertussis toxin (PTX; 0.1 µg/ml) during
the 90-min preincubation step. Staining of leukocytes with an
anti-CCR1 mAb (2D4) and the anti-CCR3 mAb (7B11) in two donors
showed no changes in eosinophil CCR1 and CCR3 expression after any
preincubation condition compared with that in cells stained immediately
after purification.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100 nM (data not shown). This may
reflect a lower level of in vivo priming in our mildly atopic donors
than those used by other studies. Monocytes, in contrast, showed
chemokine-induced shape change responses to MIP-1, MCP-1, MCP-3, and
MCP-4 only. The association between FSC in flow cytometry and cell size is well recognized, but it has also been shown that changes in this parameter can provide a measure of neutrophil polarization and agonist-induced shape change 43, 44, 45 . Neutrophil shape change in response to FMLP and PMA has been measured using flow cytometry 44, 45 , and using laser turbimetry, eosinophils have been shown to undergo a brief shape change response to platelet-activating factor (PAF) and C5a 46 . This shape change requires cytoskeletal rearrangement through the polymerization of cytoplasmic monomeric actin (G-actin) to microfilamentous actin (F-actin) 47, 48 , and eotaxin has been shown to induce eosinophil actin polymerization 49 . Eosinophil shape change in response to eotaxin was completely abolished by PTX, consistent with studies showing that eotaxin-induced actin polymerization is inhibited by pretreatment of cells with PTX 49 . In the studies by Kernen et al. the eosinophil responses to PAF and C5a were significantly, but not completely, inhibited by PTX 46 , suggesting that for some chemoattractants eosinophil shape change may be partially mediated by PTX-resistant G proteins. The response measured by the GAFS assay was abolished by pretreatment of PMNL with cytochalasin B, supporting the dependence of the response on actin polymerization 50 . The GAFS response is therefore most likely to represent a direct measure of leukocyte shape change, but may also involve contributions from changes in cell size (volume). Further studies using electron microscopy are underway to investigate the nature of leukocyte shape responses measured in the GAFS assay.
Assays investigating eosinophil responses to chemoattractants are hampered by the low numbers of these cells in the circulation and the relative complexity of their purification. The GAFS assay is sensitive, requires small numbers of cells, and exploits the characteristic autofluorescence of human eosinophils 51 , avoiding cell purification procedures that may themselves modulate leukocyte responses. The assay proved to be reliable and robust over time, but required periodic optimization of the FSC detector to maximize consistency of the results.
In contrast to eotaxin and eotaxin-2, the eosinophil response to MCP-4 showed time-dependent reversal to baseline values and a markedly bell-shaped dose-response curve. The reversal of the MCP-4-induced shape change only occurred in eosinophils; monocytes from the same donors assayed simultaneously showed shape change responses to MCP-4 that reached stable plateau levels with time and increasing agonist concentration. Eosinophil responses to both MCP-4 and eotaxin were up-regulated by preincubation of the PMNL without Ca2+ and Mg2+, although the up-regulation of responses was more marked for MCP-4 than for eotaxin. MCP-4 is thought to act on human eosinophils solely through CCR3 15, 16 . This suggests that either MCP-4 causes different eosinophil responses compared with eotaxin despite signaling through the same receptor, or that MCP-4 may modulate its own signaling through CCR3 by selective actions on an additional and as yet undescribed receptor signaling pathway. The significance of the reversal of the MCP-4-induced shape change is not clear, but may represent a potential mechanism to enhance the specificity of this chemokine in vivo, favoring eosinophil accumulation at low concentrations and monocyte accumulation at high concentrations.
Using the GAFS assay we identified three individuals whose eosinophils
showed shape change responses to MIP-1 that were equipotent to
eotaxin under the standard preincubation conditions (30 min in the
presence of Ca2+ and Mg2+). These were
designated MHR individuals. Eosinophil shape change responses to
eotaxin, RANTES, and MCP-4 were not different between MHR and other
donors. The responses to MIP-1 observed in the GAFS assay correlated
with the ability of MIP-1 to induce an intracellular calcium flux in
purified eosinophils from an MHR individual. In contrast, eosinophils
from the majority of donors showed only weak MIP-1-induced
eosinophil shape change responses and poor intracellular calcium flux
in response to this chemokine (designated MPR).
Our data showed that pretreatment of cells from MHR individuals with 10
µg/ml of a blocking anti-human CCR3 mAb 34 completely inhibited
the eosinophil responses to eotaxin, but did not inhibit eosinophil
responses to MIP-1 and only partially neutralized eosinophil shape
change responses to RANTES. Thus, these data provide evidence for the
existence of a functional shared MIP-1/RANTES receptor distinct from
CCR3 in the MHR donors that is most likely to be CCR1. When used at a
concentration of 50 µg/ml, the anti-CCR1 mAb, 2D4, blocks the
CCR1-mediated intracellular calcium flux of monocytes and the
chemotactic response of activated T cells to MIP-1 in vitro (S. Qin,
unpublished observation). In the GAFS assay, pretreatment of
cells from MHR individuals with 10 µg/ml 2D4 had no effect on their
eosinophil shape change responses to MIP-1 or eotaxin. Pretreatment
of PMNL with 50 µg/ml 2D4 caused variable suppression of eosinophil
shape change responses to both MIP-1 and, to a lesser extent,
eotaxin. A degree of nonspecific inhibition of eosinophil chemokine
responses was also seen when PMNL were pretreated with 50 µg/ml of an
isotype-matched control Ab (data not shown). The failure of the
anti-CCR1 mAb to reliably block responses in the GAFS assay is
likely to be assay and Ab concentration dependent and suggests that the
affinity of 2D4 for CCR1 may be less than the affinity of the 7B11 Ab
for CCR3.
FACS analysis of CCR1 and CCR3 expression on eosinophils in whole blood
showed high levels of CCR3 expression that were consistent between
donors and much lower levels of CCR1 expression. This is consistent
with previous data showing low levels of 125I-labeled
MIP-1 binding to human eosinophils 13, 18 . There was a higher
level of CCR1 expression on eosinophils from an MHR donor than on those
from donors whose eosinophils were poorly responsive to MIP-1.
However, even in the MHR donor the levels of eosinophil CCR1 expression
were considerably less than those of CCR3. These data additionally show
that an absence of MIP-1 responsiveness is not due to a complete
absence of CCR1 expression.
Monocyte responses to MIP-1 were similar in the MHR and MPR groups
(EC50, 0.3 vs 0.5 nM). However, it is possible that the
enhanced MIP-1 responsiveness in the eosinophils of MHR individuals
may be due to a mutation in CCR1 enhancing MIP-1 binding or
signaling, and this is the subject of further studies.
There were no clear differences in the clinical presentations of the
MHR and MPR groups. The MHR individuals were atopic nonasthmatics, and
only one of the three experienced regular symptoms to allergens
(rhinitis on exposure to house dust mites). Therefore, it is unlikely
that the MHR population represents a more severe group of atopics
showing in vivo priming to MIP-1 as has been described for IL-8
42 .
Similar to the modulation of eotaxin and MCP-4 responses, preincubation
of PMNL for 90 min without Ca2+ and Mg2+
up-regulated eosinophil MIP-1 responses in the MPR group of donors.
This up-regulation of MIP-1 responsiveness was not associated with
changes in CCR1 or CCR3 expression levels. These data together with the
immunofluorescence data reported above suggest that the MIP-1
receptor is present on eosinophils at low levels in a nonfunctional
state in the majority of individuals. Regulation of receptor
responsiveness might be mediated through receptor phosphorylation,
although receptor phosphorylation is generally thought to occur in
response to direct activation of receptors by agonist 52 . However, it
has been shown that receptors are not required to be occupied by
agonist to be phosphorylated, as phosphorylation of unstimulated
chemoattractant receptors occurs during the process of heterologous
receptor desensitization 40, 53 . Alternatively, the eosinophil
MIP-1 receptor in most individuals may be held in an inactivated
complex together with molecules such as arrestins 54 . In
IL-2-activated NK cells MIP-1 signaling is mediated via different G
proteins than RANTES, as demonstrated by a selective resistance of
MIP-1 signaling to PTX 55 ; however, in our study eosinophil
responses to MIP-1 were fully blocked by PTX.
Controversy has existed over the ability of MIP-1 to cause a
chemotactic response in human eosinophils; some groups have shown
MIP-1-induced eosinophil chemotaxis 33 , while others have not
32 . Our data suggest that MIP-1 responsiveness is likely to be a
function of both CCR1 expression levels and the regulated efficiency of
CCR1 coupling to intracellular signaling pathways. Although in our
experiments classification of donors as MHR or MPR has been consistent
over a 2-yr period, we cannot exclude the possibility that both
expression levels and coupling efficiency may vary independently within
individuals over time and with disease activity (e.g., in asthma).
Further studies of much larger populations of individuals over a long
period of time are required to investigate these possibilities fully.
Although our experiments have not investigated eosinophil chemotaxis
directly, they provide an explanation for the variations seen in
eosinophil responses by different research groups.
MIP-1 is one of several chemokines whose synthesis is up-regulated
in the human asthmatic lung, together with eotaxin and RANTES 9, 10, 30, 31 . CCR3 antagonists may prove to be valuable drugs targeting
eosinophilic allergic inflammation. However, such drugs may be of
reduced effectiveness in individuals whose eosinophils are highly
responsive to MIP-1. Furthermore, we have shown that eosinophil
responses to MIP-1 can be up-regulated in MPR individuals in vitro.
Pathways are therefore likely to exist allowing the up-regulation of
eosinophil MIP-1 responsiveness to occur in vivo; the endogenous
stimuli may be present at sites of acute inflammation associated with
asthma. Screening of individuals using functional techniques such as
the GAFS assay will be vital during recruitment of individuals for
clinical trials of novel anti-chemokine receptor therapies. Ongoing
studies should elucidate whether MHR status correlates with a clinical
phenotype in the setting of asthma and allergic disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ian Sabroe, Section of Leukocyte Biology, Divisions of Biomedical Sciences and National Heart and Lung Institute, National Heart and Lung Institute, Dovehouse St., London, United Kingdom SW3 6LY.
3 Current address: Faculty of Pharmacy, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
4 Abbreviations used in this paper: CCR, C-C chemokine receptor; MCP, monocyte chemotactic protein; MIP-1, macrophage inflammatory protein-1; GAFS, gated autofluorescence/forward scatter; FSC, forward scatter; VLA-4, very late Ag-4; PMNL, polymorphonuclear leukocytes (comprising eosinophils and neutrophils); SSC, side scatter; MHR, MIP-1-highly responsive; MPR, MIP-1-poorly responsive; PTX, pertussis toxin.
Received for publication September 8, 1998. Accepted for publication November 17, 1998.
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C. Penido, H. C. Castro-Faria-Neto, A. Vieira-de-Abreu, R. T. Figueiredo, A. Pelled, M. A. Martins, P. J. Jose, T. J. Williams, and P. T. Bozza LPS Induces Eosinophil Migration via CCR3 Signaling Through a Mechanism Independent of RANTES and Eotaxin Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 707 - 716. [Abstract] [Full Text] [PDF]
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M. J. Cotter, K. E. Norman, P. G. Hellewell, and V. C. Ridger A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic Separation Am. J. Pathol., August 1, 2001; 159(2): 473 - 481. [Abstract] [Full Text]
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C. Bandeira-Melo, K. Sugiyama, L. J. Woods, and P. F. Weller Cutting Edge: Eotaxin Elicits Rapid Vesicular Transport-Mediated Release of Preformed IL-4 from Human Eosinophils J. Immunol., April 15, 2001; 166(8): 4813 - 4817. [Abstract] [Full Text] [PDF]
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S. J. Till, L. A. Jopling, P. A. Wachholz, R. L. Robson, S. Qin, D. P. Andrew, L. Wu, J. van Neerven, T. J. Williams, S. R. Durham, et al. T Cell Phenotypes of the Normal Nasal Mucosa: Induction of Th2 Cytokines and CCR3 Expression by IL-4 J. Immunol., February 15, 2001; 166(4): 2303 - 2310. [Abstract] [Full Text] [PDF]
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A. Heinemann, A. Hartnell, V. E. L. Stubbs, K. Murakami, D. Soler, G. LaRosa, P. W. Askenase, T. J. Williams, and I. Sabroe Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling J. Immunol., December 15, 2000; 165(12): 7224 - 7233. [Abstract] [Full Text] [PDF]
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F. J. Culley, A. Brown, D. M. Conroy, I. Sabroe, D. I. Pritchard, and T. J. Williams Eotaxin Is Specifically Cleaved by Hookworm Metalloproteases Preventing Its Action In Vitro and In Vivo J. Immunol., December 1, 2000; 165(11): 6447 - 6453. [Abstract] [Full Text] [PDF]
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J. B. Domachowske, C. A. Bonville, J.-L. Gao, P. M. Murphy, A. J. Easton, and H. F. Rosenberg The Chemokine Macrophage-Inflammatory Protein-1{alpha} and Its Receptor CCR1 Control Pulmonary Inflammation and Antiviral Host Defense in Paramyxovirus Infection J. Immunol., September 1, 2000; 165(5): 2677 - 2682. [Abstract] [Full Text] [PDF]
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B. LAMKHIOUED, E. A. GARCIA-ZEPEDA, S. ABI-YOUNES, H. NAKAMURA, S. JEDRZKIEWICZ, L. WAGNER, P. M. RENZI, Z. ALLAKHVERDI, C. LILLY, Q. HAMID, et al. Monocyte Chemoattractant Protein (MCP)-4 Expression in the Airways of Patients with Asthma . Induction in Epithelial Cells and Mononuclear Cells by Proinflammatory Cytokines Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): 723 - 732. [Abstract] [Full Text] [PDF]
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M. J Leckie, S. A Bryan, J. Khan, A. Dewar, S. L Aikman, J. McGrath, D. A Okrongly, J. F Burman, P. J Barnes, and T. T Hansel Automated quantitation of circulating neutrophil and eosinophil activation in asthmatic patients Thorax, June 1, 2000; 55(6): 471 - 477. [Abstract] [Full Text]
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J. Tavernier, J. Van der Heyden, A. Verhee, G. Brusselle, X. Van Ostade, J. Vandekerckhove, J. North, S. M. Rankin, A. B. Kay, and D. S. Robinson Interleukin 5 regulates the isoform expression of its own receptor alpha -subunit Blood, March 1, 2000; 95(5): 1600 - 1607. [Abstract] [Full Text] [PDF]
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N. Zimmermann, B. L. Daugherty, J. M. Stark, and M. E. Rothenberg Molecular Analysis of CCR-3 Events in Eosinophilic Cells J. Immunol., January 15, 2000; 164(2): 1055 - 1064. [Abstract] [Full Text] [PDF]
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 V Gouon-Evans, M. Rothenberg, and J. Pollard Postnatal mammary gland development requires macrophages and eosinophils Development, January 6, 2000; 127(11): 2269 - 2282. [Abstract] [PDF]
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C. Bandeira-Melo, M. Phoofolo, and P. F. Weller Extranuclear Lipid Bodies, Elicited by CCR3-mediated Signaling Pathways, Are the Sites of Chemokine-enhanced Leukotriene C4 Production in Eosinophils and Basophils J. Biol. Chem., June 15, 2001; 276(25): 22779 - 22787. [Abstract] [Full Text] [PDF]
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I. Sabroe, M. J. Peck, B. J. Van Keulen, A. Jorritsma, G. Simmons, P. R. Clapham, T. J. Williams, and J. E. Pease A Small Molecule Antagonist of Chemokine Receptors CCR1 and CCR3. POTENT INHIBITION OF EOSINOPHIL FUNCTION AND CCR3-MEDIATED HIV-1 ENTRY J. Biol. Chem., August 18, 2000; 275(34): 25985 - 25992. [Abstract] [Full Text] [PDF]
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