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and 4 Integrin/VCAM-1 Adhesion Pathways1
*
Imperial College School of Medicine at the National Heart and Lung Institute, London, United Kingdom;
Biogen, Cambridge, MA 02142; and
Kennedy Institute of Rheumatology, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and Methods ResultsDiscussionReferences |
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4 integrin mAb, or a combination of both mAbs,
before their injection into recipient rats, the IL-4-induced cell
accumulation was inhibited by 63, 60, and 74%, respectively. Finally,
coadministration of IL-4 with the soluble TNF receptor (p55)-IgG fusion
protein significantly reduced the 111In-eosinophil
accumulation induced by the cytokine, and TNF- was detected in
IL-4-injected skin sites by both immunostaining and bioassay. Our
results demonstrate that IL-4 is a potent inducer of eosinophil
accumulation in vivo, the response being dependent on the endogenous
generation of TNF-, ß2 integrins, and 4
integrin/VCAM-1 interactions. |
Introduction |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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IL-4 is an inflammatory cytokine produced during Ag-specific immune
responses and has been implicated in allergic inflammatory disorders
such as allergic rhinitis and asthma (3, 6, 7, 8, 9, 10). It is a product of
activated CD4+ T lymphocytes of the Th2 subset and is
also released by human mast cells (11), basophils (12), and eosinophils
(3, 10). Originally described as a costimulator of B cell proliferation
(13), IL-4 is the principal cytokine involved in IgE production
(14, 15, 16). In addition, IL-4 can induce a number of other inflammatory
responses, primarily mediated via actions on endothelial cells.
Cultured endothelial cells activated with IL-4 express VCAM-1 with
little or no ICAM-1 or E-selectin expression (17, 18). This selective
induction of VCAM-1 is associated with increased adherence of
eosinophils, basophils, and lymphocytes, but not neutrophils (17, 19, 20). The reason for the lack of effect on neutrophils is explained by
the fact that the principal leukocyte ligands for VCAM-1 are the
integrins 4ß1 (VLA-4) and
4ß7, primarily expressed on the cell
surface of mononuclear cells, basophils, and eosinophils, but not
neutrophils (5, 21, 22, 23). Both 4ß1 and
4ß7 can also bind to fibronectin (24, 25),
4ß7 also acting as a ligand for another
adhesion molecule, mucosal addressin cell adhesion molecule-1 (5, 26).
The interactions of 4 integrins with VCAM-1 have also
been implicated in IL-4-stimulated endothelial cell transmigration of
eosinophils from allergic individuals (27, 28). In addition to inducing
the expression of VCAM-1 when used alone, IL-4 can also act
synergistically with IL-1 or TNF- to induce the expression of VCAM-1
in vitro (17, 18, 20, 29) and in vivo (30, 31, 32). Thus, the induction of
VCAM-1 by IL-4 has been postulated to contribute to the relatively
selective recruitment of lymphocytes and eosinophils into sites of
allergic inflammation.
In vivo, eosinophil accumulation in allergic diseases has been
associated with increased expression of IL-4 and other Th2 cytokines
(3, 6, 7, 8, 9, 10). In addition, there is both direct and indirect evidence
from a small number of animal studies indicating that IL-4 is
associated with eosinophil recruitment. In this context, i.p. or
i.d.3 injection of IL-4 has
been shown to induce selective eosinophilia in nude mice (33). Studies
addressing the in vivo effects of IL-4 have also been conducted using
the tumor-cytokine transplantation assay (34, 35). Such investigations,
which involve the IL-4 gene being transfected and expressed in
malignant cells and in turn introduced into recipient mice, have shown
that IL-4 exhibits a potent antitumor activity that is associated with
an inflammatory infiltrate comprised predominantly of eosinophils and
macrophages (34, 35). Similarly, transgenic mice overexpressing IL-4
have increased serum IgE levels and severe conjunctivitis characterized
by eosinophil infiltration (36). Further, in murine models of airway
inflammation, eosinophil infiltration was markedly suppressed in
IL-4-deficient mice (37) or following the in vivo neutralization of
IL-4 (38). The aim of our study was to extend the above findings by
investigating the ability of IL-4 to induce eosinophil accumulation in
a rat model (39) and to characterize the response. Our findings
demonstrate that, as in mice, IL-4 induces eosinophil accumulation that
is slowly developing and is not associated with a concomitant
accumulation of neutrophils. Further, our findings are the first in
vivo evidence for the involvement of endogenously generated TNF-,
and the adhesion molecules VCAM-1 (but not ICAM-1) and 4
and ß2 integrins in the IL-4-induced eosinophil
accumulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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Male Sprague Dawley cell donor rats (400–500 g) and male Sprague Dawley test rats (200–300 g) were purchased from Harlan-Olac, Oxfordshire, U.K.
Materials
Pentobarbitone sodium (Sagatal, 60 mg/ml) was purchased from May
and Baker, Dagenham, U.K. Hypnorm (0.315 mg/ml fentanyl citrate and 10
mg/ml fluanisone) was purchased from Janssen Pharmaceutical, Grove,
U.K. Hypnovel (5 mg/ml midazolam hydrochloride) was purchased from
Roche Products, Welwyn Garden City, U.K.
111InCl3 (10 mCi/ml in pyrogen-free 0.04 N
hydrochloric acid) and 125I-HSA (20 mg albumin per ml of
sterile isotonic saline, 50 µCi/ml) were purchased from Amersham
International, Amersham, U.K. BSA, 2-mercaptopyridine
N-oxide, control mAb MOPC-21 (mouse myeloma IgG), saponin,
and PAF were purchased from Sigma Chemical, Dorset, U.K. Horse serum,
sterile HBSS, HEPES, and Tyrode’s salt solution were purchased from
Life Technologies, Paisley, U.K. Percoll was purchased from Pharmacia
Fine Chemicals, Uppsala, Sweden. Pyrogen- and preservative-free heparin
sodium (5000 U/ml) was purchased from Pabyrn Laboratories, Greenford,
U.K. Chromotrope 2R was from BDH, Poole, U.K. The anti-rat CD18 mAb
WT.3 (mouse IgG1) (40) was purchased from AMS Biotechnology, Oxon, U.K.
The 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 previously described (41). Recombinant rat IL-4
(IL-4) was from Dr. D. Mason, MRC Cellular Immunology Unit, Sir
Williams Dunn School of Pathology, University of Oxford, Oxford, U.K.
This preparation of IL-4 contained 1.4 ng/ml of endotoxin (i.e.,
maximum amount of endotoxin injected into skin sites was 0.14 ng/site)
as assayed using a Limulus amebocyte lysate assay
(BioWhittaker, Walkersville, MD). The anti-human 4
integrin mAb HP2/1 (mouse IgG1), recognizing rat 4 (42),
the anti-rat VCAM-1 mAb 5F10 (mouse IgG2a) (43), and rhTNF-
(TNF-) were from Biogen, Cambridge, MA. The soluble TNF- receptor
(p55)-IgG fusion protein (TNFR-IgG) and a control chimeric Ab (cSF25),
reactive to a human panadenocarcinoma Ag, were from Dr. B. J.
Scallon and Dr. J. Ghrayeb, Centocor Inc., Malvern, PA.
Purification and 111In-labeling of rat peritoneal eosinophils
Rat peritoneal eosinophils were elicited and purified as previously described (39). Briefly, rats were injected i.p. with 5 ml of horse serum and killed 1 to 2 days later by CO2-induced asphyxia. Peritoneal cells were collected by lavage with 30 ml of heparinized saline (10 U/ml) and purified by centrifugation over a three-layer discontinuous Percoll-HBSS gradient (60%/65%/75%). The fractionated cell population was used only when the eosinophil purity, as determined by Kimura staining, was >90%. The predominant contaminating cell type was mononuclear, and a major exclusion criterion was the presence of neutrophils.
The purified eosinophils were radiolabeled with 111In as
previously described (39). Briefly, the cells were incubated with
111InCl3 (
100 µCi in 10 µl) chelated
with 2-mercaptopyridine N-oxide (40 µg in 0.1 ml of 50 mM
PBS, pH 7.4) for 15 min at room temperature. The labeled leukocytes
were washed three times and resuspended (1 x 107
cells/ml) in HBSS solution, pH 7.4, containing cell-free citrated rat
plasma to a final concentration of 10%.
Measurement of 111In-eosinophil accumulation and edema formation in rat skin
Leukocyte infiltration and edema formation in the rat dorsal skin were simultaneously measured using the local accumulation of i.v. injected 111In-labeled cells and 125I-HSA as previously described (39). 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 was shaved. IL-4, freshly prepared from stock solution in Tyrode’s solution with low endotoxin BSA (0.1%), was injected i.d. (100 µl/site), in duplicate into the back skin, 48, 24 or 0 h before the labeled cells were injected. Eosinophils (5 x 106 cells in 0.5 ml of HBSS) mixed with 125I-HSA (2.5 µCi/animal) were then injected i.v. via a tail vein. After 5 min, other agents under investigation were injected i.d. into the previously clipped back skin. At the end of a 4-h test period, the animals were reanesthetized, and a cardiac blood sample was collected. The animals were then killed by an overdose of sodium pentobarbitone, the back skin was removed, and the injection sites were punched out with a 17-mm-diameter punch. Skin, blood, and plasma samples were counted in an automatic 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 accumulation in each skin site in terms of the number of labeled leukocytes, for 5 x 106 cells injected per rat. 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.
Histology
IL-4 (5000 U/site)- or Tyrode’s solution-injected rat skin sites were punched out as described above. The skin sections were then fixed in 10% phosphate-buffered formalin for 24 h, routinely processed, and embedded in paraffin wax. Sections of 5 µm were stained with Chromotrope 2R (44).
Immunostaining of skin sites for TNF-
IL-4 (5000 U/site)- or Tyrode’s solution-injected rat skin
sites were punched out as descibed above and immediately embedded in
OCT embedding matrix (Cell Path, Hemel Hempstead, U.K.), snap-frozen in
isopentane, prechilled in liquid nitrogen, and stored at -70°C until
use. Sagittal sections (6 µm thick) were stained for cell-associated
TNF- as previously described (45). Briefly, sections were air-dried
for 30 min at room temperature, fixed for 15 min in ice-cold 4%
paraformaldehyde, and then washed in HEPES-buffered balanced salt
solution, supplemented with 0.1% saponin. Endogenous peroxidase
activity was blocked by immersing the sections in 1% hydrogen peroxide
and 0.1 M sodium azide dissolved in balanced salt solution/saponin for
30 min at room temperature in the dark. For detection of TNF-, the
sections were incubated overnight (at 4°C) with a cytokine-specific
Ab (PharMingen, San Diego, CA). Control staining was performed in
parallel using a species- and isotype-matched myeloma protein. The
sections were then washed and incubated for 1 h at room
temperature with a biotinylated secondary Ab (Vector, Burlingame, CA),
and Ab-biotin conjugates were detected with an
avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC,
Vector). A color reaction was developed using diaminobenzidine
(Peroxidase Substrate Kit, Vector). In parallel sections, the mouse mAb
R-73 (Serotec, Oxford, U.K.), directed against the ß TCR complex
(TCR), was used as a primary Ab, and a staining procedure similar to
that described above was used. The specificity of the methodology for
TNF- staining was verified by the complete inhibition of staining
with rTNF-. To localize and identify TNF--producing cells, entire
tissue sections were examined using an Olympus BH2 microscope and
analyzed by image analysis (AnalySIS, Soft Imaging System GmbH,
Munster, Germany). TNF--positive cells in each sample were
calculated based on the mean number of positive cells in three
semiserial sections per each specimen.
Preparation and bioassay of rat skin sites for TNF-
activity
Rat skin sites were punched out, snap frozen in liquid nitrogen,
cut into small pieces and subjected to three cycles of 15-s
homogenizations in HBSS (Ultra-Turrax T25, Janke and Kunkel, Staufen,
Germany). The samples were then centrifuged twice (10 min at 3000
x g and 20 min at 13,000 x g), and
the supernatants were stored at -70°C until assayed for TNF-
activity. Supernatant samples (350–250-µl volumes) were serially
diluted and assayed on WEHI 164 cells for TNF--mediated cytotoxicity
as previously described (46). A TNF- standard curve was incorporated
into each assay and used to express the results in terms of picograms
of TNF- in each skin site.
Statistical analysis
Results are expressed as the mean ± SEM for n animals, where each datum unit is the average of responses in duplicate sites. Data were analyzed by two-way ANOVA of log-transformed data, and statistical significance was determined with the Newman-Keuls procedure for repeated comparisons. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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To determine the time course of IL-4-induced
111In-eosinophil accumulation, IL-4 at a dose of 3000
U/site or Tyrode’s solution was injected i.d. into the clipped back
skin of recipient animals at 48, 24, or 0 h before the i.v.
injection of 111In-eosinophils. After a further 4 h in
vivo test period, the animals were killed, and the responses were
quantified (Fig. 1
). Injection of IL-4
i.d. 48 h before the measurement period did not cause the
accumulation of 111In-eosinophils. However, there was a
marked increase in cell accumulation in sites injected with the
cytokine 0 or 24 h before the measurement period, both protocols
eliciting significantly greater accumulation than that detected in
Tyrode’s solution-injected sites (Fig. 1). The 24-h time point was
chosen for all subsequent experiments.
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At the 24-h time point, i.d. administration of IL-4 caused a
dose-dependent accumulation of 111In-eosinophils in
rat skin, with significant responses being achieved at 1000, 3000, and
5000 U/site (Fig. 2A).
Within the same dose range, however, IL-4 was totally inactive with
respect to stimulation of edema formation (Fig. 2B).
Further, histologic examination of IL-4-injected skin sites
demonstrated the ability of this cytokine to induce the accumulation of
eosinophils, as well as mononuclear cells, but not the accumulation of
neutrophils (Fig. 3).
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The following experiments were conducted to investigate the
adhesive pathways involved in the IL-4-induced
111In-eosinophil accumulation. Administration i.v. of
an anti-VCAM-1 mAb (5F10, 2 mg/kg) significantly inhibited the
111In-eosinophil accumulation induced by IL-4 (Fig. 4A). The responses
elicited by 1000, 3000, and 5000 U/site of the cytokine, injected at
-24 h, were significantly reduced by 73, 57, and 49%, respectively,
in 5F10-treated rats (Fig. 4A). A larger dose of the
Ab (5 mg/kg) induced a greater inhibition of the IL-4-induced response,
e.g., the 111In-eosinophil accumulation induced by 3000
U/site of IL-4 was inhibited by 79.3 ± 10.1%
(n = 3 pairs). In contrast to the effects seen
with the anti-VCAM-1 mAb, an anti-ICAM-1 mAb (1A29, 5 mg/kg)
had no significant effect on the IL-4-induced
111In-eosinophil accumulation (Fig. 4B).
Both Abs, however, inhibited the 111In-eosinophil
accumulation induced by PAF (10-10 moles/site, at
0 h).
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4 and
ß2 integrins on IL-4-induced 111In-eosinophil
accumulation
Rat eosinophils bound the anti-human 4
integrin mAb HP2/1 and the anti-rat ß2 integrin mAb
WT.3 with a saturating concentration of 2 µg/ml/106
cells, as assessed by flow cytometry (data not shown). Based on these
results, the two Abs were used at this concentration to pretreat
111In-eosinophils in vitro before their i.v. injection into
recipient rats. Briefly, after their final wash, the radiolabeled cells
were divided into four aliquots: aliquot 1, cells treated with a
control Ab, MOPC-21 (4 µg/ml/106 cells); aliquot 2, cells
treated with HP2/1 (2 µg/ml/106 cells); aliquot 3, cells
treated with WT.3 (2 µg/ml/106 cells), and aliquot 4,
cells treated with both HP2/1 and WT.3. With all four aliquots, the
111In-eosinophils were treated with the Abs for 20 min at
room temperature followed by their i.v. administration into recipient
animals. The radiolabeled cells were then allowed to circulate for
4 h as before. As described above, IL-4 was administered i.d.
24 h before the cells were injected.
Figure 5 shows the effects of the in
vitro pretreatment of 111In-eosinophils with the
anti-4 mAb, the anti-ß2 mAb, or
the combination of both Abs on 111In-eosinophil
accumulation in rat skin induced by IL-4. Both HP2/1 and WT.3
significantly inhibited the IL-4-induced 111In-eosinophil
accumulation by 63 and 60%, respectively. Although the combination of
both Abs caused a larger reduction in the IL-4-elicited
111In-eosinophil accumulation (74%), this was not
significantly different from the inhibition obtained with either Ab
alone. In addition, in a second set of experiments, i.v. HP2/1 (3.5
mg/kg) inhibited the 111In-eosinophil accumulation induced
by IL-4 (5000 U/site) by 78.1 ± 8.1% (n =
4 pairs of rats).
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receptor (p55)-IgG fusion protein on
IL-4-induced 111In-eosinophil accumulation
Coadministration of TNFR-IgG (100 µg/site), but not the control
chimera cSF25, together with TNF- totally inhibited the
111In-eosinophil accumulation induced by TNF- (Fig. 6). Further, the coinjection of the
soluble TNF- receptor with IL-4 resulted in a significant
suppression of the 111In-eosinophil accumulation elicited
by this cytokine (50% inhibition (Fig. 6A)). In
contrast, TNFR-IgG had no effect on responses induced by two other
inflammatory mediators, PAF and LTB4 (Fig. 6B), indicating its selectivity for certain stimuli.
Unpublished studies have shown that TNFR-IgG also has no effect on the
leukocyte accumulation induced by IL-1 (results not shown).
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in IL-4-injected skin
sites
To extend the above studies with the TNFR-IgG, IL-4-injected rat
skin sites were bioassayed for TNF--like activity. Figure 7 shows that TNF--like activity was
detected in rat skin sites injected with IL-4 (5000 U/site) in a
time-dependent manner. In contrast, no significant levels of TNF-
activity were detected in skin sites injected with PAF
(10-10 mol/site) or LPS (3 ng/site) (results not
shown). To localize the source of this TNF-, IL-4-injected skin
sites were immunostained for the cytokine. TNF--positive cells were
found in all sections prepared from IL-4-injected skin sites (Fig. 8) where as no positive cells were
detected in samples prepared from Tyrode’s solution-injected sites
(not shown). In the IL-4-injected samples, the TNF--positive cells
were predominantly infiltrating leukocytes found in the dermal region,
and very few cells were observed s.c. In addition, in some regions,
TNF--positive cells were found associated with the vessel wall (Fig. 8). Analysis of the TNF--expressing cells, according to morphology
and cell size, indicated that 86 ± 8% of the cells were
lymphocytes while 14 ± 5% of the cells were macrophage-like
cells. The association of TNF- with T lymphocytes was subsequently
confirmed with the use of an anti-TCR mAb in sequential sections,
which indicated that 30% of the total number of TNF--positive
cells were stained with the anti-TCR mAb.
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Discussion |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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4 and
ß2 integrins, VCAM-1, and ICAM-1 in the IL-4-induced
eosinophil accumulation. Further, by using a functional blocker of
TNF- (a soluble TNF receptor (p55)-IgG fusion protein) and by
bioassaying and immunostaining IL-4-injected skin sites for TNF-, we
have shown a role for endogenously generated TNF- in the
IL-4-induced eosinophil accumulation. The in vivo model that we have
used for these studies involves measuring the accumulation of
111In-labeled eosinophils in rat skin, a model previously
developed to characterize the 111In-eosinophil accumulation
induced by IL-1ß and TNF- (39, 47).
Injection of IL-4 i.d. induced a slowly developing accumulation of
111In-eosinophils, with significant levels being detected
within the measurement periods of 0 to 4 h and 24 to 28 h
(studied in detail), but not 48 to 52 h. Within the 24- to 28-h
time frame, the 111In-eosinophil accumulation induced by
IL-4 was dose dependent. Further, histologic examination of
IL-4-injected skin sites demonstrated the accumulation of eosinophils
and mononuclear cells but not the accumulation of neutrophils. The
IL-4-induced selective recruitment of eosinophils and mononuclear
cells, as opposed to neutrophils, observed in our studies agrees with
the in vivo findings of Moser et al. (33) and Tepper et al. (34, 35, 36).
In the former study, IL-4 was injected i.d. or i.p. into mice, and cell
infiltration was quantified histologically. In the latter studies,
Tepper et al. used two approaches to investigate IL-4-induced responses
in vivo. One model involved studying the phenotypes of a series of
transgenic mice with varying levels of IL-4 expression in B or T
lymphocytes (36), and the other involved transplanting IL-4-secreting
malignant cells into recipient mice (34, 35). In both models, IL-4
generation was associated with the development of eosinophil-rich
inflammatory lesions. Although the in vivo time course of eosinophil
accumulation induced by exogenously administered IL-4 has not been
previously reported, our results are in close agreement with the
reported time course of IL-4-induced endothelial cell adhesiveness for
eosinophils in vitro (19, 27). Further, the selective accumulation of
eosinophils induced by IL-4 is consistent with in vitro findings where
adherence to, and transmigration across, IL-4-treated cultured
endothelial cells was specific for eosinophils as opposed to
neutrophils (19, 27, 28, 33). This relatively selective interaction of
eosinophils with IL-4-activated endothelial cells has been attributed
to the fact that IL-4 induces the expression of VCAM-1 but not other
endothelial cell adhesion molecules such as ICAM-1 or E-selectin (18, 19, 48), and neutrophils do not express high levels of the VCAM-1
ligands, 4 integrins (21, 22, 23).
Although the functional involvement of 4
integrins/VCAM-1 adhesion pathways in eosinophil adhesion to
IL-4-activated cultured endothelial cells has been well documented (19, 27, 28, 33), there is as yet no in vivo evidence for this interaction.
To investigate this possibility, the effect of an anti-rat VCAM-1
mAb, 5F10, on the eosinophil accumulation induced by IL-4 was tested.
This Ab dose-dependently inhibited the 111In-eosinophil
accumulation induced by IL-4 (3000 U/site), resulting in 57 and 80%
inhibition of the response at 2 and 5 mg/kg of the mAb, respectively.
Furthermore, an anti-4 integrin mAb, HP2/1, known to
cross-react with rat eosinophils, also inhibited the IL-4-induced
111In-eosinophil accumulation. An inhibitory effect was
achieved with this Ab whether it was used to pretreat the
111In-eosinophils in vitro (63% inhibition) or given i.v.
(78% inhibition). Both the anti-VCAM-1 mAb and the
anti-4 integrin mAb also partially suppressed the
PAF-induced 111In-eosinophil accumulation. However, an
anti-rat ICAM-1 mAb, 1A29, although again partially attenuating the
response to PAF, had no effect on the 111In-eosinophil
accumulation induced by IL-4. These results strongly implicate an
important role for the integrins 4ß1
and/or 4ß7 and the endothelial cell
adhesion molecule VCAM-1, but not ICAM-1, in the eosinophil
accumulation induced by IL-4. Our results are consistent with the in
vitro studies cited above and are supported by the findings of Fukuda
et al. showing a correlation between IL-4 levels and eosinophil numbers
in the bronchoalveolar lavage fluid of allergic asthmatics and the
expression of VCAM-1, but not ICAM-1 or E-selectin, on vascular
endothelium in bronchial mucosal biopsies (49). More recently, the
IL-4-induced leukocyte accumulation in skin in cynomolgus monkeys has
also been correlated with an increased expression of VCAM-1 but not of
ICAM-1 or E-selectin (50).
Of importance, in contrast to our results with i.v. anti-ICAM-1 mAb, we found that the in vitro pretreatment of radiolabeled eosinophils with an anti-rat ß2 integrin mAb (WT.3) inhibited the 111In-eosinophil accumulation induced by IL-4. Because ICAM-1 is an important ligand for ß2 integrins, these results may appear to be in conflict, but are in fact in agreement with some in vitro findings. Eosinophil adhesion to and transmigration through IL-4-activated cultured endothelial cell monolayers, although ICAM-1 independent, can be partially inhibited by anti-ß2 integrin mAbs (19, 27). A number of explanations may account for our in vivo results. One possible reason may be differences in the ability of the two Abs to act as functional blockers. However, a difference in efficacy is unlikely to account for the divergent effects seen on the IL-4-induced eosinophil accumulation as both Abs suppressed the responses induced by eosinophil chemoattractants such as PAF and eotaxin (data not shown). A more likely explanation is that in response to IL-4, ß2 integrins may be interacting with endothelial cell ligand(s) other than ICAM-1, as has been suggested in other studies (51, 52, 53, 54, 55). The accumulation of neutrophils into sites of inflammation in ICAM-1-deficient mice also suggests the possible existence of other ligands for ß2 integrins (56). The full complement of cell adhesion and activating molecules expressed on endothelial cells following activation with IL-4 has yet to be determined. Interestingly, Yao et al. have recently reported the expression of P-selectin on IL-4-activated endothelial cells (57).
The slowly developing time course of IL-4-induced 111In-eosinophil accumulation suggests that, in addition to inducing the expression of adhesion molecules, the response elicited by IL-4 may also be mediated by the endogenous generation of secondary inflammatory mediators. In this context, a number of molecules have been implicated in the IL-4-induced inflammatory responses. Stem cell factor, a mast cell differentiation and activation factor, is reportedly released from IL-4-activated mouse alveolar macrophages and can mediate eosinophil accumulation in a murine model of allergen-induced airway inflammation (58). Murine resident peritoneal macrophages stimulated with IL-4 have also been shown to express mRNA for the chemokine C10 (59), and IL-4 has been implicated in the generation of MCP-1 both in vitro and in vivo (50, 60). Interestingly, the eosinophil-specific CC chemokine eotaxin (61, 62) is induced locally following the transplantation of IL-4-secreting tumor cells in recipient mice (63), suggesting a likely role for this chemokine in IL-4-induced eosinophil recruitment.
In addition to the above cytokines/chemokines, TNF- has also been
implicated in the inflammatory responses elicited by IL-4. Increased
levels of both TNF- and IL-4 have been detected in bronchoalveolar
lavage from asthmatics (7, 8, 9, 64) and in murine models of allergic lung
inflammation where they appear to have key roles in the induction of
leukocyte recruitment and chemokine generation (38, 65, 66). Further,
functional blockade of TNF- completely inhibits the IL-4-induced
expression of VCAM-1 on murine isograft microvascular endothelial cells
(31). In addition to the above, there is much evidence showing a
synergistic interaction between these cytokines. In vitro, IL-4 can
synergistically and selectively enhance the expression of VCAM-1 on
endothelial cells stimulated with TNF- (18, 29). In addition, the
combination of these two cytokines has been shown to enhance
endothelial cell adhesiveness for T cells but not neutrophils, raising
the possibility that whereas TNF- is nonselective in terms of
leukocyte adhesion to endothelial cells, in combination with IL-4 it
can induce the selective recruitment of lymphocytes as opposed to
neutrophils (17). Further, in a primate model, s.c. administered IL-4,
in combination with subinflammatory doses of TNF-, augmented VCAM-1
expression on microvascular endothelial cells and caused selective T
cell infiltration (30).
To investigate the role of endogenously generated TNF- in the
IL-4-induced eosinophil accumulation, we tested the effect of TNFR-IgG.
TNFR-IgG, although totally inhibiting the 111In-eosinophil
accumulation induced by TNF-, when coinjected with IL-4 also
significantly suppressed the 111In-eosinophil accumulation
induced by this cytokine (50%). This effect was selective given that
TNFR-IgG had no effect on the responses elicited by PAF or
LTB4. In addition, TNF--like bioactivity was detected in
IL-4-injected, but not PAF-injected, skin sites in a time-dependent
manner. IL-4-injected skin sites immunostained for TNF- indicated
that the cytokine was predominantly associated with infiltrating
mononuclear leukocytes. Our results provide the first line of evidence
directly implicating endogenously generated TNF- in IL-4-induced
eosinophil accumulation and indicate the cellular source of the
cytokine as infiltrating mononuclear leukocytes.
The findings of this study suggest that the IL-4-induced early response
of eosinophil accumulation (0–4 h) is mediated primarily by the direct
effect of IL-4 on venular endothelial cells via the expression of
VCAM-1. This stage of the response may also involve the endogenous
generation of other inflammatory mediators such as the CC chemokine
eotaxin. Indeed, in a separate study, we have found that eotaxin
message can be detected in IL-4-injected rat skin sites as early as
4 h postinjection (M.-J. Sanz et al., manuscript in preparation).
The IL-4-induced eosinophil accumulation detected at the later time
points (24–28 h), as well as being mediated by the cellular components
discussed above, may also be driven by the infiltration of other
leukocyte types, specifically monocytes and lymphocytes, via the
generation of TNF-. Although in vitro studies have indicated that
IL-4 causes a down-regulation of TNF- generation from monocytes and
suppresses lymphocyte function (67), the in vivo interaction of IL-4,
TNF-, and leukocytes has yet to be fully understood.
Hence, it is possible that at sites of allergic inflammation, TNF-
generated from inflammatory cells in response to IL-4 may synergize
with IL-4 to induce the expression of VCAM-1, providing a mechanism by
which a specific adhesion pathway is activated, selective for certain
subsets of leukocytes. The synergistic interaction of these cytokines
means that very small levels of IL-4 and TNF- may be required to
induce endothelial cell activation resulting in both increased
expression and prolonged appearance of VCAM-1 on the endothelium (29, 31). Therefore, although the ability of exogenously administered
TNF- to induce neutrophil accumulation has been well documented
(68, 69, 70), the levels of TNF- generated in response to IL-4 may be
too small to induce the accumulation of neutrophils, resulting in the
selective accumulation of eosinophils. Previous studies have shown that
both exogenously administered and endogenously generated TNF- can
induce eosinophil accumulation in vivo (47, 71).
In summary, we have shown that IL-4 is a potent inducer of eosinophil
accumulation in rat skin and that its effect is selective for
eosinophil and mononuclear cell recruitment as opposed to neutrophils.
Further, the IL-4-induced eosinophil accumulation is primarily mediated
through 4 integrin/VCAM-1 interactions, adding to the
increasing list of inflammatory reactions mediated by this adhesion
pathway (72, 73, 74). In addition, there is a ß2
integrin-dependent, but ICAM-1-independent, component to the eosinophil
recruitment elicited by IL-4, suggesting a role for other endothelial
cell ligands. Our results also suggest a role for endogenously
generated TNF- in the IL-4-induced eosinophil accumulation, further
illustrating the complexity of the cellular and molecular events
mediating the selective accumulation of eosinophils into sites of
inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sussan Nourshargh, Cardiovascular Medicine Unit, Imperial College School of Medicine at the National Heart and Lung Institute, Hammersmith Hospital, Du Cane Road, London, W12 ONN U.K. E-mail address:
3 Abbreviations used in this paper: i.d., intradermal; PAF, platelet-activating factor; LTB4, leukotriene B4; TNFR-IgG, soluble TNF- receptor (p55)-IgG fusion protein; HSA, human serum albumin.
Received for publication December 11, 1997. Accepted for publication February 3, 1998.
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References |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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) at 48, 24, and 0 h before i.v.
injection of the 111In-eosinophils. The cells were allowed
to circulate for 4 h after which the animals were killed, and
responses were measured as described in Materials and
Methods. 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 levels detected in Tyrode’s
solution-injected sites is indicated by ** p <
0.01.
)) and control mAb MOPC-21
(2 mg/kg i.v. (
)). B, Effect of the anti-ICAM-1 mAb
1A29 (5 mg/kg i.v. (
