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4 and ß2 Integrin-Dependent Eosinophil Accumulation in Rat Skin In Vivo: Delayed Generation of Eotaxin in Response to IL-41
*
Leukocyte Biology Centre, Biomedical Sciences Division, Imperial College School of Medicine, London, United Kingdom;
Leukosite Inc., Cambridge, MA 02142;
Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan;
§
Department of Immunology, University of Liverpool, Liverpool, United Kingdom; and
¶
Biogen Inc., Cambridge, MA 02142
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin mAbs significantly inhibited the eosinophil
accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%,
respectively. Further, when 111In-eosinophils were
pretreated in vitro with anti-4 integrin or
anti-ß2 integrin mAbs, or with a combination of both
mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and
68%, respectively. Eosinophil accumulation induced by intradermal
IL-4, but not that induced by TNF- or leukotriene B4,
appeared to be mediated in part by endogenously generated eotaxin.
Anti-eotaxin Abs significantly inhibited (54%) the later phases
(24–28 h) but not the early phase (0–4 h) of the response to IL-4.
This was consistent with eotaxin mRNA expression peaking at 18 h
after IL-4 injection. Our findings show that human eotaxin is a potent
inducer of eosinophil accumulation in vivo, this response being
dependent on 4 integrin/vascular cell adhesion
molecule-1 and ß2 integrin/intercellular adhesion
molecule-1 adhesion pathways. Further, the eosinophil accumulation in
response to IL-4 is partly mediated by endogenously generated eotaxin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eotaxin, a CC chemokine originally purified from the bronchoalveolar
lavage fluid of allergen-challenged guinea pigs, is a potent and
selective eosinophil chemoattractant that is active both on guinea pig
and human cells (3, 4). Guinea pig (5, 6), mouse (7, 8), human (9, 10),
and rat (11) eotaxin have now been cloned. Human eotaxin exhibits a
similar high potency and selectivity for human eosinophils, inducing
the elevation of intracellular free calcium concentrations, chemotaxis,
actin polymerization, production of reactive oxygen species, and
Mß2 (CD11b/CD18) up-regulation in
vitro (9, 10, 12, 13). Although eotaxin is constitutively expressed at
low levels in most human organs, marked up-regulation in protein or
message has been detected in the epithelium and submucosa of nasal
polyp tissues (9) and in the intestine of patients with inflammatory
bowel diseases (10), where there is a clear eosinophil infiltration.
The major cellular sources of eotaxin are thought to be the epithelium,
the endothelium, and activated infiltrating leukocytes such as
monocytes and eosinophils (9, 10, 14). Human eotaxin has been
demonstrated by histologic examination to induce eosinophil
accumulation in monkey skin (9). We found that human eotaxin was
inactive on guinea pig eosinophils, both in vitro and in vivo (data not
shown) despite the observation that guinea pig eotaxin is highly active
on human eosinophils (4). We then found that 125I-human
eotaxin bound to rat eosinophils (data not shown). Therefore, we have
used a previously described rat skin model (15, 16) to examine
inflammatory responses induced by human eotaxin in vivo.
The initial interaction between leukocytes and the vascular
endothelium, namely rolling, is a transient and reversible adhesion
mainly mediated by the selectins and their carbohydrate counterligands
(17, 18), although recently the 4 integrins,
4ß1 (very late activation Ag-4) and
4ß7, have also been implicated in this
process (19, 20). Rolling is thought to result in the exposure of
leukocytes to chemoattractants, such as chemokines, presented on the
surface of endothelial cells (21). The chemokines then trigger a firm
adhesion of leukocytes that is thought to be mediated through the
interaction of integrins with their endothelial counterreceptors:
vascular cell adhesion molecule
(VCAM4)-1 for
4 integrins (for review, see 22 and intercellular
adhesion molecule (ICAM)-1 and ICAM-2 for ß2 integrins
(23, 24). These adhesive interactions serve to facilitate the
subsequent transendothelial migration of leukocytes along
chemoattractant gradients (25, 26).
Interactions of 4 integrins with VCAM-1 and fibronectin
can mediate eosinophil accumulation at sites of inflammation because
eosinophils, but not normally neutrophils, express 4
integrins on their surface (22), and mAbs against the 4
integrin subunit or VCAM-1 inhibit eosinophil accumulation in several
in vivo models of allergic and nonallergic inflammation (16, 27, 28, 29).
Further, the recent cloning of the human eotaxin receptor, CCR3, and
its high expression on eosinophils (30, 31) provide a molecular basis
for the eosinophil selectivity of eotaxin. Hence, all these findings
have led us to hypothesize that the combination of the chemoattractant
activity of eotaxin together with its possible role in integrin
activation might determine the selective eosinophil traffic observed at
sites of eosinophilic inflammation. Our findings show that human
eotaxin is a potent inducer of eosinophil accumulation in vivo, and
that this response is dependent on 4 and
ß2 integrins and their endothelial cell counterligands.
Further, we provide evidence that endogenously generated eotaxin is
involved in eosinophil accumulation induced by IL-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Sprague Dawley cell donor rats (400–500 g) and male Sprague Dawley in vivo assay rats (200–300 g) were purchased from Harlan-Olac, Bicester, Oxfordshire, U.K.
Materials
Pentobarbitone sodium (Sagatal, 60 mg/ml) was purchased from May
and Baker, Dagenham, Essex, U.K. Hypnorm (0.315 mg/ml fentanyl citrate
and 10 mg/ml fluanisone) was from Janssen Pharmaceutical, Grove,
Oxford, U.K. Hypnovel (5 mg/ml midazolam hydrochloride) was from Roche
Products, Welwyn Garden City, U.K.
111InCl3 (10 mCi/ml in pyrogen-free 0.04 N
hydrochloric acid), 125I-human serum albumin (HSA; 20 mg of
albumin per ml of sterile isotonic saline, 50 µCi/ml),
[-32P]dCTP, Multiprime DNA labeling system, and Hybond
N hybridization transfer membranes were from Amersham International,
Amersham, U.K. BSA (<0.1 ng of endotoxin per mg), 2-mercaptopyridine
N-oxide, control mAb MOPC-21 (mouse myeloma IgG1),
platelet-activating factor (PAF), glycogen, and Freund’s complete and
Freund’s incomplete adjuvants were from Sigma Chemical, Poole, Dorset,
U.K. Sterile HBSS, HEPES, Tyrode’s salt solution, and TRIzol were from
Life Technologies, Paisley, U.K. Percoll and protein A-Sepharose were
from Pharmacia Fine Chemicals, Uppsala, Sweden. Pyrogen- and
preservative-free heparin sodium (5000 U/ml) was from Pabyrn
Laboratories, Greenford, U.K. Restriction enzymes and RNA m.w. markers
were from Promega, Southampton, U.K. Fura-2 acetoxymethyl ester was
from Cambridge Bioscience, Cambridge, U.K. Leukotriene B4
(LTB4) was from Cascade Biochemicals, Reading, U.K.
Recombinant human TNF- was from Biogen, Cambridge, MA. Recombinant
rat IL-4 was from Serotec, Oxford, U.K. Synthetic human eotaxin (9) was
from Leukosite, Cambridge, MA.
Anti-rat ß2 mAb WT.3 (mouse IgG1) was from AMS
Biotechnology, Oxford, U.K. and was generated as described elsewhere
(32). Anti-rat ICAM-1 mAb 1A29 (mouse IgG1) was generated by
immunization of mice with endothelial cells obtained from high venules
of rat lymph nodes as described in detail in a previous study (33). The
anti-human 4 integrin mAb HP2/1 (mouse IgG1),
recognizing rat 4 (34), and the anti-rat VCAM-1 mAb
5F10 (mouse IgG2a) (16) were from Biogen.
Purification of rat leukocytes
Rat leukocytes were elicited and purified as previously
described (15, 16). Eosinophils were used only when their purity, as
determined by Kimura staining, was >94%. The predominant
contaminating cell type was mononuclear and a major exclusion criterion
was the presence of neutrophils. When these cells were compared by FACS
analysis to eosinophils immunostained in whole blood, there was no
significant difference in the expression of 4
integrins and L-selectin (16). Mononuclear cells (>97% pure at the
upper interface) were prepared from the same donors and gradients as
neutrophils (>98% pure at the lower interface).
Elevation of intracellular calcium
Rat eosinophils, neutrophils, and mononuclear leukocytes (107 cells/ml in Ca2+/Mg2+-free PBS containing 0.1% BSA) were loaded with fura-2 acetoxymethyl ester (2.5 µM, 30 min at 37°C). After two washes, cells were resuspended at 106 cells/ml in Ca2+/Mg2+-free PBS containing 10 mM HEPES/0.25% BSA/10 mM glucose, pH 7.4. Aliquots were dispensed into quartz cuvettes and the external Ca2+ concentration adjusted to 1 mM with CaCl2. Changes in fluorescence were monitored at 37°C using an LS50 fluorescence spectrophotometer (Perkin-Elmer, Beaconsfield, U.K.) at excitation wavelengths 340 nm and 380 nm, and emission wavelength 510 nm. [Ca2+]i levels were calculated using the ratio of the readings at the two excitation wavelengths and a Kd for Ca2+ binding at 37°C of 224 nM (35). Responses were monitored for 3 min, and data are expressed as maximal increase in [Ca2+]i over the basal levels.
111In-labeling of rat eosinophils and neutrophils
Rat eosinophils or neutrophils were radiolabeled with 111In as previously described (15, 16). Briefly, the cells (1–2 x 107) were incubated with 111InCl3 (approximately 100 µCi chelated with 40 µg 2-mercaptopyridine-N-oxide in 0.1 ml of 50 mM sodium phosphate, pH 7.4) for 15 min at 20°C. The labeled leukocytes were washed three times and resuspended (1 x 107 cells/ml) in HBSS, pH 7.4, containing cell-free citrated rat plasma to a final concentration of 10%. The final cell suspension normally carried approximately 60% of the total radioactivity used, and 5 x 106 111In-cells were injected into each recipient rat.
Measurement of 111In-leukocyte accumulation and edema formation in rat skin
Leukocyte infiltration and edema formation were simultaneously measured using the local accumulation of i.v.-injected 111In-labeled cells and 125I-HSA, as previously described (15). Briefly, rats were anesthetized with a mixture of Hypnorm (0.1 ml/rat) and Hypnovel (0.1 ml/rat), injected i.p., and their dorsal skin shaved. 111In-eosinophils or 111In-neutrophils (5 x 106 cells mixed with 2.5 µCi 125I-HSA) were then injected i.v. via a tail vein. Five minutes later, the agents under investigation were injected (100 µl/site, in Tyrode’s salt solution containing 0.1% BSA) into the back skin. At the end of a 4-h test period, or 1 h for the time course experiment, the animals were reanesthetized and a cardiac blood sample was collected for determination of cell and plasma radioactivity. The animals were then killed by an overdose of sodium pentobarbitone, the back skin removed, and the injection sites punched out with a 17-mm-diameter punch. Skin, blood cell pellet, and plasma samples were counted in a Cobra Auto Gamma counter (Canberra Packard, Pangbourne, U.K.) and counts were cross-channel corrected for the two isotopes. The 111In count per cell was determined and used to express eosinophil or neutrophil accumulation in each skin site in terms of the number of labeled leukocytes. Edema formation at each site was expressed as microliters of plasma by dividing skin sample 125I counts by 125I counts in 1 µl of plasma.
In vitro pretreatment of 111In-eosinophils with
anti-4 mAb HP2/1 and anti-ß2 mAb
WT.3
After their last wash, 111In-eosinophils were divided into four groups for pretreatment with mAbs: 1) MOPC-21 (10 µg/5 x 106 cells/0.5 ml), 2) HP2/1 (5 µg/5 x 106 cells), 3) WT.3 (5 µg/5 x 106 cells), and 4) WT.3 + HP2/1 (each at 5 µg/5 x 106 cells). These mAb concentrations were determined by indirect immunostaining and FACS analysis to be saturating. After incubation for 20 min at 20°C, the cells were injected i.v. into recipient animals.
Generation of anti-human eotaxin Ab
Rabbits were immunized with human synthetic eotaxin (0.2 mg/animal) emulsified in Freund’s complete adjuvant. Booster injections in incomplete adjuvant were given at 3 and 6 wk and the animals bled out 12 days after the last boost. The IgG fraction of the antiserum was purified by binding to protein A-Sepharose; elution with 0.1 M glycine-HCl, pH 3.0; collection into sodium phosphate, pH 7.4, to limit exposure to acid; and dialysis against saline. The IgG was adjusted to the original volume of serum, filtered, and stored at -20°C in aliquots.
Northern blot analysis
A full-length rat eotaxin cDNA of 980 bp, isolated from an inflamed lung cDNA library (11), was excised from the vector pBKCMV (Stratagene, Cambridge, U.K.) by restriction digestion with BamHI and XhoI. To control for RNA loading, a full-length rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA of 1272 bp was used.
IL-4 (5000 U/100 µl) and Tyrode’s/0.1% BSA vehicle were injected
into the skin of three rats 24, 18, 8, and 4 h before killing with
an anesthetic overdose. The skin sites were quickly punched out and
frozen at -80°C. Total RNA was extracted using TRIzol according to
the manufacturer’s instructions. RNA samples (10 µg) were separated
on 1% agarose gels, containing 2.2% formaldehyde, transblotted onto
nylon membranes, and fixed by UV irradiation. Membranes were hybridized
with the rat eotaxin cDNA probe (labeled using
[-32P]dCTP and Multiprime kits). Membranes were washed
at a final stringency of 0.1x SSC, 0.1% SDS, 55°C (2 x 20
min) and autoradiographed at -70°C. The membranes were stripped,
rehybridized using the 32P-labeled rat GAPDH cDNA probe,
and washed at the same high stringency. Autoradiographs were assessed
by laser densitometry. Data are expressed as the ratio of eotaxin to
GAPDH mRNA: the sample in each rat with the highest ratio being given
an arbitrary value of one and other samples expressed relative to
this.
Statistical analysis
Results are expressed as the mean ± SEM for n animals or cell preparations. For the in vivo experiments, each datum unit is the average of responses in duplicate skin sites. Data were analyzed by two-way ANOVA of log-transformed data and statistical significance determined with the Newman-Keuls procedure for repeated comparisons. A p value <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human eotaxin induced a rapid and dose-dependent increase in
[Ca2+]i in rat eosinophils with a
significant effect at 1, 10, and 30 nM, the maximal effect being at 10
nM (Fig. 1
). Responses peaked within
30 s and returned to basal levels (76.1 ± 2.2 nM
Ca2+i) by approximately 2 min. The nonselective
chemoattractant, PAF, used as a positive control, caused a similar
increase in [Ca2+]i when assayed at 10 nM
(Fig. 1).
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Effect of human eotaxin on 111In-eosinophil accumulation and edema formation in rat skin in vivo
Using a 4-h in vivo time period, intradermal (i.d.) administration
of human eotaxin caused a dose-dependent accumulation of
111In-labeled eosinophils in rat skin with significant
responses being achieved at 10 and 100 pmol/skin site (Fig. 2A). Intradermal PAF at
100 pmol/skin site also induced a significant eosinophil accumulation
(Fig. 2A). In contrast, human eotaxin did not induce
any edema formation (Fig. 2B) or
111In-neutrophil accumulation (data not shown) while PAF
(100 pmol/site, used as a positive control) induced significant
responses in each case. Histologic examination of the skin injected
with human eotaxin (100 pmol/site) demonstrated the presence of an
eosinophil infiltrate, with no neutrophil infiltrate, after a 4-h in
vivo test period in animals not injected i.v. with labeled cells (data
not shown).
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Human eotaxin at a dose of 10 pmol/skin site or Tyrode’s/BSA
vehicle was injected i.d. into different skin sites 240, 120, 60, 30,
and 0 min before the i.v. injection of
111In-eosinophils. After a 1-h in vivo test period,
the animals were killed and the responses quantified. Significant cell
accumulation in response to the chemokine was detected within the first
2 h (i.e., at 0–60, 30–90, and 60–120 min) but not at later
time points (Fig. 3).
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4 integrin mAb HP2/1 on responses in
rat skin induced by human eotaxin
To study the adhesion pathways involved in eotaxin-induced
eosinophil accumulation in vivo, the effects of mAbs directed against
different adhesion molecules were determined. Abs were i.v.
administered, 15 min before injecting the labeled cells, at 3.5 mg/kg
for anti-4 integrin mAb (HP2/1) and 5 mg/kg for
anti-ICAM-1 mAb (1A29), anti-VCAM-1 mAb (5F10), and the control
mAb (MOPC-21). In a previous study, these i.v. doses of mAbs were found
to be sufficient to give the maximum attainable inhibitory effects on
TNF--induced eosinophil accumulation (16). Once the cells were
injected, human eotaxin at 10 and 100 pmol/skin site was i.d.
administered and responses allowed to develop for 4 h. Anti-ICAM-1
mAb significantly reduced the eosinophil accumulation elicited by
eotaxin (Fig. 4A), the
response to the top dose being reduced by 73%. Similarly, the
anti-VCAM-1 and anti-4 integrin mAbs also
significantly inhibited the eosinophil accumulation induced by human
eotaxin; at 100 pmol eotaxin/skin site, responses were reduced by 43
and 67%, respectively (Fig. 4, B and C).
These inhibitory effects could not be explained by a reduction in the
circulating 111In-eosinophils numbers, as the
anti-4 integrin mAb had no significant effect on the
circulating numbers, while the anti-ICAM-1 and the anti-VCAM-1
mAbs increased circulating 111In-eosinophils (by 46 and
61%, respectively).
|
4 integrin mAb HP2/1 and
anti-ß2 integrin mAb WT.3 on responses in rat skin
induced by human eotaxin
Anti-4 integrin mAb HP2/1 (16) and
anti-ß2 integrin mAb WT.3 (data not shown) bind to
rat eosinophils. The effect of in vitro pretreatment of
111In-eosinophils with these mAbs, before i.v. injection of
the cells, on their accumulation in eotaxin-injected skin sites is
shown in Figure 5. Anti-4
integrin and anti-ß2 integrin mAbs significantly
inhibited 111In-eosinophil accumulation in response to
eotaxin by 52 and 49%, respectively. Although the combination of both
Abs appeared to cause a larger reduction in the chemokine-elicited
responses (68%), this effect was not significantly different from the
effects of either of the Abs alone.
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Northern blot analysis showed a time-dependent eotaxin mRNA
expression at sites of IL-4 injection (5000 U/skin site). Eotaxin
message was virtually undetectable in vehicle-injected sites but was
increased, beginning at 4 h and reaching a peak at approximately
18 h, after IL-4 injection (Fig. 6).
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(Fig. 7). IL-4 (5000 U/skin site) was
coinjected with control rabbit IgG or anti-human eotaxin IgG at two
different time points: -24 h and 0 h, each in the same animal so
that each rat acted as its own internal control. Labeled cells were
then injected i.v. and responses allowed to develop for 4 h.
Anti-human eotaxin IgG reduced the 111In-eosinophil
accumulation induced by IL-4 injected at -24 h (i.e., the 24- to 28-h
response) in all rats tested (n = 8,
p < 0.05) but had no effect on the 0- to 4-h
eosinophil accumulation response to IL-4 (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize the eosinophil accumulation elicited by human eotaxin,
we have used an in vivo assay system to measure
111In-eosinophil accumulation and edema formation in
rat skin (15, 16). Preliminary experiments showed that
125I-human eotaxin bound to rat eosinophils in vitro (data
not shown). This binding caused a rapid and dose-dependent increase in
intracellular calcium levels (Fig. 1
). In contrast, human eotaxin
failed to elicit changes in intracellular calcium concentrations in
either rat neutrophils or rat mononuclear cells. This demonstrates the
eosinophil selectivity of eotaxin in the rat, in agreement with
previous in vitro studies in other species (8, 9, 10, 36). Next, we
demonstrated that human eotaxin elicits a potent and selective (Fig. 2)
eosinophil accumulation in rat skin; no neutrophil accumulation or
edema formation was seen. The eosinophil accumulation was a rapid
event, significant responses occurring within the first hour (Fig. 3).
Taken together with the in vitro data on calcium mobilization in
isolated cells, this rapid eosinophil accumulation suggests a direct
effect of eotaxin on eosinophils in vivo. Chemokines such as eotaxin
are thought to be presented on the microvascular endothelium and
stimulate adhesion (21).
The adhesive mechanisms that regulate chemokine-induced accumulation of
leukocytes in vivo remain largely uncharacterized. As part of the
present study, we have investigated the roles of the ß2
integrins/ICAM-1 and 4 integrins/VCAM-1 adhesion
pathways in the process of eosinophil accumulation elicited by eotaxin
(Figs. 4 and 5). Pretreatment of 111In-eosinophils in vitro
with a saturating concentration of a neutralizing
anti-ß2 integrin mAb, before their administration
into the recipient animals, significantly suppressed the
eotaxin-induced 111In-eosinophil accumulation. Further, the
i.v. administration of a neutralizing anti-ICAM-1 mAb also caused
an inhibition of this response. These results suggest that
eotaxin-induced leukocyte accumulation is at least partly mediated via
the direct effects of the chemokine on the leukocytes causing
up-regulation or activation of ß2 integrins in vivo.
ß2 integrins can then mediate the firm attachment of the
eosinophils to venular endothelial cells via basally expressed ICAM-1.
Previous studies have demonstrated the importance of the
ß2 integrins or ICAM-1 in eosinophil accumulation in
other models of inflammation (37, 38, 39, 40). We have found that eotaxin
up-regulates the expression of ß2 integrins on rat
peripheral blood eosinophils in vitro (K. Nagai et al., unpublished
observations). If this up-regulation also occurs in vivo, the newly
expressed ß2 integrins will not be blocked by the in
vitro pretreatment of the eosinophils with mAb. This would explain why
we found that blocking the existing ß2 integrins in vitro
(Fig. 5) was less effective than blocking the ICAM-1 counterligand in
vivo (Fig. 4).
Neutralizing mAbs directed against 4 integrins or VCAM-1
also suppressed the eosinophil accumulation induced by i.d. eotaxin.
The anti-4 integrin mAb was effective at suppressing
this response whether it was used to pretreat the
111In-eosinophils in vitro or given i.v. These results
suggest that, in addition to activating ß2 integrins, the
eotaxin-induced eosinophil accumulation may be mediated via
4 integrins although the mechanism by which eotaxin may
activate 4 integrins is still unknown. VCAM-1, one of
the principal endothelial cell ligands for 4 integrins,
is generally considered to be expressed only on cytokine-activated
endothelial cells (22). However, as an anti-VCAM-1 mAb was
effective at attenuating the rapid eosinophil accumulation induced by
eotaxin, our results suggest the presence of low levels (that may be
undetectable by immunostaining) of basally expressed VCAM-1 on venular
endothelial cells that are sufficient to mediate the accumulation of
eosinophils. These results are in agreement with our previous findings
in which we reported that an anti-4 integrin mAb
could inhibit the eosinophil accumulation induced by rapidly acting
chemoattractants such as LTB4 and
C5adesArg (27).
The pretreatment of 111In-eosinophils with a combination of
anti-4 integrin and anti-ß2
integrin mAbs did not cause a significantly larger level of inhibition
than that seen with either mAb alone. This differs from the additive
effect seen with two similar Abs, which completely inhibited the
eosinophil accumulation induced in guinea pig airways in response to
i.v. Sephadex particles (29). It is possible that 4 and
ß2 integrins may be involved in different but sequential
stages of the eotaxin-induced eosinophil migration, namely rolling and
firm adhesion, respectively. In this respect, there is in vivo evidence
for the involvement of 4 integrins in eosinophil rolling
(19), as well as in the subsequent phase of firm adhesion, whereas
ß2 integrins are not thought to contribute to eosinophil
rolling.
Recent in vitro studies have addressed the mechanisms involved in
chemokine-mediated leukocyte adhesion and transendothelial cell
migration. Eosinophil transmigration across unstimulated endothelial
cell monolayers induced by RANTES can be virtually abolished by mAbs
directed against ß2 integrins (41). Although this effect
was reported to occur without an apparent increase in ß2
integrin expression on the leukocyte surface, recent reports have
demonstrated that RANTES, monocyte chemoattractant protein (MCP)-3, and
eotaxin can stimulate the up-regulation of
Mß2 integrin (CD11b/CD18) on human
eosinophils (13, 42). In addition, RANTES and MCP-3 can trigger a
strong and prolonged adhesion of human eosinophils to ICAM-1, this
effect being primarily mediated by direct activation of
ß2 integrins (42). Eosinophils can also adhere to either
VCAM-1 or fibronectin upon stimulation with chemokines (25, 26, 42, 43). This event was shown to be rapid and transient, and occurred
without apparent changes in 4ß1 integrin
expression on the leukocyte surface (26, 42), although it was
correlated with f-actin polymerization (26, 42), uropod-like formation,
and asymmetrical distribution of very late activation Ag-4 (26). Hence,
the above in vitro studies largely support the present in vivo findings
and suggest that the eotaxin-induced eosinophil migration may be
mediated by changes in the expression, affinity states, or signaling
pathways of 4 and ß2 integrins.
IL-4 is a Th2 cytokine that selectively induces VCAM-1 expression on
endothelial cells with little or no effect on ICAM-1 or E-selectin
expression (44, 45, 46). This feature of IL-4 has been associated with the
selective adhesion and transmigration of eosinophils, as opposed to
neutrophils (47, 48, 49, 50), across the vascular endothelium mediated by
4 integrins on the eosinophil surface (22). In vivo,
i.p. or i.d. injection of IL-4 induces selective eosinophil
accumulation in nude mice and rats (49, 51). In agreement with in vitro
studies, the IL-4-induced eosinophil accumulation in vivo appears to be
4 integrin/VCAM-1 dependent (51). Besides inducing
VCAM-1 expression, IL-4 can exert eosinophil accumulation through the
endogenous generation of secondary inflammatory mediators. In other
systems, IL-4 has been implicated in the release of stem cell factor
from alveolar macrophages (52), in the increased expression of C10 in
murine resident peritoneal macrophages (53), and in the synthesis and
secretion of MCP-1 by cultured HUVECs (54).
Eotaxin message has been detected after transplantation of
IL-4-secreting tumor cells in mice, an effect that occurs within 6
h and persists for 7 days (7). We conducted a series of experiments to
detect the possible involvement of eotaxin generation in eosinophil
accumulation induced by i.d.-injected IL-4 in the rat. Firstly,
Northern blot analysis showed that eotaxin message began to increase at
4 h in IL-4-injected sites and was still present at 24 h
(Fig. 6). Secondly, we used a neutralizing anti-human eotaxin Ab to
block part of the response to IL-4. 111In-eosinophil
accumulation observed 24 to 28 h after, but not 0 to 4 h
after, IL-4 injection was significantly suppressed when the cytokine
was coinjected with the Ab (Fig. 8). These effects of anti-eotaxin
are consistent with the mRNA data, which suggest the likely presence of
eotaxin protein during the 24- to 28-h 111In-eosinophil
accumulation period but little or no eotaxin release during the 0- to
4-h period. Hence, we conclude that endogenous eotaxin generation
partly mediates the later phases of eosinophil accumulation induced by
IL-4. This response to eotaxin may, in turn, be dependent on the
up-regulation of VCAM-1 on the endothelial cell surface in response to
IL-4, as discussed above.
Th2 cytokines appear to play a key role in regulating eotaxin-induced eosinophil accumulation, since IL-5 has been shown to cooperate with eotaxin to induce eosinophil infiltration in the skin and in the lungs by mobilizing eosinophils from the bone marrow (10, 14, 55), and eosinophil infiltration induced by IL-4 can be partly mediated through eotaxin generation, as discussed above. Indeed, in two mouse models of Ag-induced pulmonary eosinophilia, it has recently been shown that elimination of T cell accumulation results in a reduction of eosinophil recruitment associated with eotaxin down-regulation (56, 57).
In conclusion, we have shown that human eotaxin is a potent and selective inducer of eosinophil accumulation in rat skin and provide the first in vivo evidence that eosinophil accumulation induced by chemokines can be abrogated by mAbs directed against both leukocyte and endothelial cell surface adhesion molecules. We have also demonstrated that the secondary generation of endogenous eotaxin is involved in the later phases of eosinophil accumulation in response to IL-4.
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Footnotes |
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2 Present address: Dr. Maria-Jesus Sanz, Departamento de Farmacologia, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot (Valencia), Spain.
3 Address correspondence and reprint requests to Dr. Peter Jose, Leukocyte Biology Centre, Imperial College School of Medicine, Dovehouse Street, London SW3 6LY. U.K. E-mail address:
4 Abbreviations used in this paper: VCAM, vascular cell adhesion molecule; PAF, platelet-activating factor; LTB4, leukotriene B4: ICAM, intercellular adhesion molecule; i.d., intradermal; HSA, human serum albumin; MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication May 12, 1997. Accepted for publication December 9, 1997.
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) were given at
different time points before i.v. injection of the
111In-eosinophils. Cell accumulation during a 1-h period
was measured. Results are expressed as number of
111In-eosinophils per site per 5 x 106
cells injected and presented as mean ± SEM for four rats. A
significant difference from vehicle-injected sites is indicated by *
p < 0.05, ** p < 0.01.
