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Immunopharmacology Laboratory, Departamento de Farmacologia and
Departmento de Patologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; and
Department of Pathology, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the cytokine stem cell factor (SCF) was shown to activate the adhesion of eosinophils to fibronectin and vascular cell adhesion molecule 1 in vitro (19). Moreover, SCF may induce the activation of eosinophils indirectly via the release of eosinophil chemoattractants from intermediate cell types. For example, fibroblasts have been shown to produce SCF, which in turn activates mast cells to produce the eosinophil-active chemokine eotaxin (20). Similarly, pretreatment of mouse bone marrow-derived mast cells with SCF induced a dose-dependent increase in LTB4 (21). Thus, if produced early in an allergic reaction, SCF could not only stimulate eosinophils directly, but also participate locally in the activation of tissue cells (e.g., mast cells), which in turn would induce the recruitment of eosinophils. However, little is known about the ability of this cytokine to participate in the recruitment of eosinophils in vivo.
The present study evaluates whether the intrapleural (i.pl.) injection of SCF induces eosinophil recruitment in vivo and whether endogenous SCF is involved in mediating eosinophil recruitment in response to Ag challenge in sensitized mice. In addition, we investigated whether the effects of SCF on eosinophil migration are direct or dependent on the local release of the lipid mediator LTB4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c mice (18–22 g) were used throughout these experiments and were housed in a temperature-controlled room with access to water and food ad libitum.
Drug and reagents
Recombinant murine SCF was obtained from PeproTech (London, United Kingdom). SCF was initially resuspended in distilled water and diluted further in PBS (pH 7.4) containing 0.01% BSA. The endotoxin level was <0.1 ng/µg of SCF (maximal injection into the peritoneal cavity of contaminating LPS was 0.01 ng) and was much lower than the dose of endotoxin needed to induce eosinophil migration in the model (>50 ng/cavity; data not shown). BSA, OVA, and control rabbit serum were purchased from Sigma (St. Louis, MO). The LTB4 antagonist CP105,696 was a gift from Pfizer (Sandwich, U.K.).
Anti-SCF Ab-SCF Ab
Anti-SCF Abs were prepared by immunizing rabbits with recombinant murine SCF as described previously (20). The IgG portion of the serum of hyperimmune or preimmune rabbits was purified over a protein A column (Pierce, Rockford, IL) and stored at -20°C in PBS until use.
Sensitization
Animals were immunized with OVA adsorbed to aluminum hydroxide gel as described previously (22). Briefly, mice were injected s.c. on days 1 and 8 with 0.2 ml of a solution containing 100 µg of OVA and 70 µg of aluminum hydroxide (Reheiss, Dublin, Ireland). This sensitization procedure was accompanied by a significant increase in total IgE as assessed by ELISA (at 1/100 dilution, OD readings were 0.088 ± 0.005 and 0.209 ± 0.049 for naive and immunized animals, respectively) and OVA specific as assessed by a 24 h-fixation period passive cutaneous anaphylaxis in rat dermis (OVA-specific IgE titer, 1/80). The immunization schedule used also resulted in blood eosinophilia (control animals, 0.3 ± 0.2 x 105 eosinophils/ml; immunized animals, day 7, 1.5 ± 0.5 x 105 eosinophils/ml; day 14, 1.0 ± 0.5 x 105 eosinophils/ml, n = 4).
Leukocyte migration into the pleural cavity induced by OVA or SCF
Seven to 8 days after the last immunization, the animals were anesthetized and Ag (OVA, 0.1–10 µg/pleural cavity) was injected i.pl. SCF (1–100 ng/pleural cavity) was injected in naive mice. Animals were killed at different times (4, 24, 48, or 72 h) after the i.pl. injection of the stimuli and the cells present in the pleural cavity were harvested by injecting 2 ml of PBS. Total cell counts were performed in a modified Neubauer chamber using Turk’s stain, and differential cell counts were performed on cytospin preparations stained with May-Grunwald-Giemsa using standard morphologic criteria to identify cell types. The results are presented as the number of cells per cavity.
Treatment with anti-SCF and LTB4 receptor antagonist
To evaluate the role of endogenous SCF, each animal was injected with a dose of 100 µg of purified anti-SCF IgG or control IgG i.v. 30 min before Ag or SCF challenge. The LTB4 receptor antagonist CP105,696 (23) was administered i.p. 60 min before challenge at a dose of 3 mg/kg.
RT-PCR
To investigate the expression of SCF mRNA in pleural fluid leukocytes, immunized animals were injected with Ag and pleural fluid washes were conducted just before challenge and at 1, 2, 4, 8, 24, and 48 h later. Five animals were used for each time point. The fluid recovered from the animals in each group was pooled and centrifuged at 1200 x g. To the cell pellet, 0.5 ml of Trizol (Life Technologies, Grand Island, NY) was added and total RNA was extracted according to the instructions of the manufacturer. One microgram of total RNA was then reverse transcribed by the addition of 2.5 U RNasin (Promega, Madison, WI), 2.5 mM dNTPs (Boehringer Mannheim, Mannheim, Germany), 0.1 M DTT (Life Technologies), Moloney murine leukemia virus RNase H-reverse transcriptase buffer (Life Technologies), 25 ng oligo(dt) oligonucleotides (Boehringer Mannheim), and 200 U Moloney murine leukemia virus RNase H-reverse transcriptase (Life Technologies) in 20 µl total volume. The reaction proceeded for 1 h at 37°C and was terminated by boiling for 5 min after the addition of 175 µl H2O. Five microliters of cDNA was used for amplification in a 25-µl PCR reaction containing 2.5 mM dNTPs (Pharmacia, Piscataway, NJ), a 0.2 mM concentration of the 3'and 5'external primers, 2.5 mM MgCl2; 1x GeneAmp PCR buffer, and 5 U Taq DNA polymerase (Promega). The SCF primers used in the reactions were: SCF (38 cycles/358 bp), 5'-CAC TCA GCT TGA CTA CTC TT and 3'-GTC ATT CCT AAG GGA GCT GG and the constitutive gene HPRT (30 cycles/162 bp) primers were 5'-GTT GGA TAC AGG CCA GAC TTT GTT G and 3'-GAT TCA ACT TGC GCT CAT CTT AGG C. PCR conditions for SCF primers were executed as follows: 95°C, 3 min, 94°, 1 min, 52° C, 1 min (first cycle), 72°C, 2 min, 94°C, 1 min (37 cycles), 72° C, 7 min (first cycle). PCR products and m.w. markers were run on 6% polyacrylamide gel and stained with silver nitrate (24). Data were analyzed in a densitometer.
ELISA for LTB4
Frozen supernatants obtained from pleural cavity washes of control animals and at 2, 8, and 24 h after SCF or 2 and 8 h after OVA challenge were centrifuged at 10,000 x g for 10 min and brought to room temperature. LTB4 in the samples were determined using a specific LTB4 detection kit (R&D Systems, Minneapolis, MN) according to instructions of the manufacturer.
Statistical analysis
All results are presented as the means ± SEM. Normalized data were analyzed by one-way ANOVA, and differences between groups were assessed using the Student-Newman-Keuls posttest. A p value < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The i.pl. injection of SCF (10–100 ng/cavity) induced a
dose-dependent recruitment of eosinophils after 48 h (Fig. 1
A). SCF (100
ng/cavity)-induced eosinophil recruitment was first detected 4 h
after injection and peaked at 24 h (Fig. 1B). There was
significant eosinophil recruitment at 48 h but the effects had
waned by 72 h (Fig. 1B). In addition to recruiting
eosinophils, SCF induced a significant recruitment of neutrophils early
after injection (4 h) and mononuclear cells at a later stage (at 24 and
48 h, see Table I).
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The i.pl. injection of Ag (OVA) in sensitized mice induced a
dose-dependent recruitment of eosinophils after 48 h (Fig. 2A). In contrast, OVA
challenge of naive animals did not induce significant eosinophil
recruitment at 48 h (naive animals: PBS, 0.4 ± 0.05; OVA, 1
µg/cavity, 0.3 ± 0.2; immunized animals: PBS, 0.4 ± 0.2;
OVA, 1 µg/cavity, 2.9 ± 0.7 x 105
eosinophils/cavity, n = 5). Eosinophil recruitment
after OVA (1 µg/cavity) challenge was indistinguishable from
background at 4 h and peaked after 24–48 h (Fig. 2B).
The number of eosinophils had dropped to baseline levels by 72 h
(Fig. 2B). Ag challenge of sensitized mice also induced a
significant early recruitment of neutrophils, peaking at 4 h and
dropping rapidly to background levels by 24 h, and a late
recruitment of mononuclear cells which was maximal at 24 h (Table II).
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). SCF expression was significant from
to 2 to 8 h after Ag challenge (Fig. 3). There was no detectable
expression of SCF mRNA in naive mice challenged with OVA or immunized
mice challenged with PBS (Fig. 3). We then evaluated the effect of the
neutralization of endogenous SCF on the migration of eosinophils in the
allergic reaction. For these experiments, OVA was used at the dose of 1
µg/cavity and eosinophil recruitment was assessed 48 h after
challenge. Pretreatment of animals with a purified anti-SCF
polyclonal Ab (100 µg of IgG/animal) abrogated the eosinophil (Fig. 4A) and mononuclear cell (data
not shown) recruitment induced by SCF. Similarly, the number of
eosinophils accumulated in the pleural cavity of sensitized mice after
OVA challenge was virtually abolished by anti-SCF pretreatment
(Fig. 4B). The effects of anti-SCF pretreatment were not
specific for eosinophils, as mononuclear cell recruitment induced by
OVA in immunized animals was effectively inhibited by pretreatment with
the polyclonal Ab (PBS, 3.1 ± 1.0 x
105; OVA, 11.5 ± 2.5 x
105; OVA + anti-SCF, 2.4 ± 1.1 x
105 mononuclear cells/cavity, n = 6,
p < 0.05). Because neutrophils migrate earlier in the
allergic reaction, we assessed the effects of anti-SCF pretreatment
on the influx of neutrophils 4 h after Ag challenge. At this early
time point, the anti-SCF Ab had little effect on neutrophil
recruitment (PBS, 0.03 ± 0.01 x 105;
OVA, 12.1 ± 1.7 x 105; OVA +
anti-SCF, 10.0 ± 0.6 x 105
neutrophils/cavity, n = 6).
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The next series of experiments were designed to investigate
whether SCF induced the recruitment of eosinophils in our model via the
endogenous release of LTB4. The i.pl. injection
of SCF induced a time-dependent increase in the levels of
LTB4 in pleural wash supernatants (Fig. 5). Maximal LTB4
levels were detected from 2 to 8 h and the levels were back to
basal by 24 h (Fig. 5). In agreement with the ability of SCF to
induce LTB4, pretreatment of mice with the
LTB4 antagonist CP105,696 (3 mg/kg) before the
i.pl. injection of SCF diminished SCF-induced eosinophil recruitment by
84% (Fig. 6A). Similarly, the
LTB4 antagonist partially inhibited SCF-induced
mononuclear cell influx at 48 h (PBS, 1.3 ± 0.3 x
105; SCF, 100 ng, 5.8 ± 0.5 x
105; SCF + CP105, 696, 4.0 ± 0.6 x
105 mononuclear cells/cavity, n = 5,
p < 0.05) and neutrophil influx at 4 h (PBS,
0.1 ± 0.03 x 105; SCF, 100 ng,
2.3 ± 0.5 x 105; SCF + CP105, 696,
0.7 ± 0.3 x 105 neutrophils/cavity,
n = 5, p < 0.05).
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B, pretreatment of animals with
CP105,696 inhibited the eosinophil recruitment following OVA challenge
by 78%. The effects of the LTB4 antagonist were
not specific for eosinophils as the mononuclear cell recruitment
48 h after Ag challenge of sensitized mice was also inhibited by
CP105,696 (PBS, 3.6 ± 1.1 x 105; OVA,
8.2 ± 1.6 x 105; OVA + CP105, 696,
3.4 ± 0.7 x 105 mononuclear
cells/cavity, n = 5, p < 0.05). In contrast
to its effects on eosinophil and mononuclear cell recruitment,
CP105,696 had little effect on neutrophil recruitment 4 h after Ag
challenge (OVA, 12.1 ± 1.7 x 105; OVA
+ CP105, 696, 9.8 ± 1.7 x 105
neutrophils/cavity, n = 5). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments were designed to investigate whether the i.pl. administration of SCF induced the migration of eosinophils. Our data demonstrate that SCF induced a dose- and time-dependent recruitment of eosinophils that was maximal 24 h after administration of 100 ng/cavity. Because SCF has been previously shown to activate human peripheral blood eosinophils directly via a functional c-kit receptor (19), experiments were conducted to investigate whether the effects of SCF were direct on the eosinophil or indirect via the release of the lipid mediator LTB4. Indeed, in vitro studies have shown that SCF stimulates tissue cells, especially mast cells, to release lipid-derived mediators (25, 26, 27, 28), including LTB4 (21). In this study, we demonstrate that there was a significant increase in the levels of LTB4 in the pleural cavity of mice following the administration of SCF. Not only were LTB4 levels elevated, but also the SCF-induced eosinophil accumulation was effectively blocked by pretreatment of the animals with a LTB4 receptor antagonist. This is in agreement with other studies demonstrating an important role for LTB4 in mediating the recruitment of eosinophils following the in vivo administration of a range of inflammatory stimuli (10, 11, 12). A direct effect for LTB4 in mediating the recruitment of eosinophils via surface LTB4 receptors has been previously demonstrated in guinea pig skin (17, 18) and murine eosinophils do possess functional LTB4 receptors (6, 29). The cellular target in which SCF is acting to release LTB4 that consequently drives the recruitment of eosinophils is not known. However, as mentioned above, the ability of SCF to induce the activation of mast cells in vitro (21, 25, 26, 27, 28) and in vivo (30) may underlie the effects observed. Thus, our data demonstrated that the in vivo administration of SCF induces the migration of eosinophils indirectly via the release of LTB4. Interestingly, the SCF-induced eosinophil migration may have functional consequences since the administration of SCF into the lungs of normal or allergic mice is accompanied by significant airway hyperactivity (31).
As demonstrated in Table I, SCF was not specific for eosinophils
because this cytokine also induced an early recruitment of neutrophils
(at 4 h) and a late mononuclear cell recruitment (peaking at
24 h). It was, thus, of interest to evaluate whether the ability
of SCF to induce the recruitment of these leukocytes was also dependent
on the local release of LTB4. Similar to the
effects on eosinophil migration, the LTB4
antagonist, CP105,696, significantly blocked both the early neutrophil
recruitment and the late mononuclear cell recruitment. These results
are consistent with the rapid rise in LTB4 levels
in the pleural cavity following SCF injection (see Fig. 5) and the
ability of LTB4 to activate neutrophils and
mononuclear cells. Thus, it appears that the ability of SCF to induce
leukocyte recruitment in the pleural cavity is largely related to its
ability to release LTB4.
The i.pl. administration of Ag to sensitized animals induced a time- and dose-dependent recruitment of eosinophils. Moreover, a significant expression of SCF mRNA in response to Ag challenge was observed following Ag challenge and preceding the maximal increase in eosinophil numbers in the pleural cavity. Interestingly, pretreatment with an anti-SCF polyclonal Ab, at a dose which effectively blocked the effects of SCF itself, abrogated the migration of eosinophils following Ag challenge. These results suggest an essential role for the endogenous release of SCF in the recruitment of eosinophils in an allergic reaction in the mouse. These results are in good agreement with our previous findings demonstrating a role for SCF in the migration of eosinophils following Ag challenge in murine lung (32). Together, our data are suggestive of an important early role for SCF in determining eosinophil recruitment following the administration of Ag. The mechanisms underlying such an in vivo effect of SCF are under active investigation in our laboratories. However, in vitro data demonstrate that SCF is capable not only of activating mast cells directly, but also of priming mast cells for IgE-dependent and IgE-independent release of a range of different inflammatory mediators (28, 33, 34). As expected from its effects on SCF-induced responses, pretreatment with a LTB4 receptor antagonist significantly blocked the eosinophil migration induced by Ag challenge in sensitized mice (around 80% inhibition). These results are in good agreement with the ability of OVA challenge to raise the i.pl. levels of LTB4 and support an essential role of LTB4 in driving the local eosinophil recruitment.
Similar to the effects of locally injected SCF, the i.pl. administration of Ag to sensitized mice induced not only eosinophil migration, but also significant early neutrophil recruitment (at 4 h) and late mononuclear cell recruitment (peaking at 24 h). We then investigated whether neutrophil and mononuclear cell recruitment in the allergic reaction were also dependent on the local release of SCF and/or LTB4. Pretreatment with CP105,696 or anti-SCF Ab significantly inhibited mononuclear cell recruitment, suggesting that, akin to eosinophil recruitment, the influx of mononuclear cells is dependent on the local release of SCF and LTB4. These results are consistent with the ability of SCF to induce mononuclear cell recruitment in a LTB4-dependent manner. Interestingly, anti-SCF or CP105,696 pretreatment had no significant effect on the number of neutrophils recruited to sites of allergic inflammation at 4 h. One possibility to explain these results lies with the kinetics of the generation of both SCF mRNA and LTB4 protein which, although already detected at 2 h after challenge, peaked later (at 8 h). Thus, although SCF can induce the recruitment of neutrophils in a LTB4-dependent manner, our results suggest that other inflammatory mediators may be released locally and mediate the recruitment of neutrophils following allergen challenge of immunized mice.
Together, our data favor the hypothesis that the local release of SCF following Ag challenge may activate and/or prime mast cells for IgE-mediated release of inflammatory mediators, especially LTB4, in the pleural cavity of mice. The mediators released in turn drive the recruitment of eosinophils and other cell types. Inasmuch as inhibition of the function of SCF in vivo may reduce the migration of eosinophils to sites of allergic inflammation, a SCF-based therapy may be a relevant principle in the treatment of allergic diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mauro M. Teixeira, Departamento de Farmacologia, Immunopharmacology Laboratory, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627, Belo Horizonte, Minas Gerais, 31270-901 Brazil.
3 Abbreviations used in this paper: LTB4, leukotriene B4; SCF, stem cell factor; i.pl., intrapleural.
Received for publication September 20, 1999. Accepted for publication February 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Br. J. Pharmacol. 125:1228.[Medline]
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