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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T-cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up-regulated by a number of agonists, including complement-dependent factors (C3b/iC3b) and interferon-γ (IFN-γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil-specific agonists. We analysed RANTES mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum-coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN-γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2-microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN-γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil-active cytokines, interleukin-3 (IL-3), IL-4, IL-5 and granulocyte–macrophage colony-stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN-γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN-γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN-γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The infiltration of eosinophils into the airways is a major feature of asthmatic inflammation. Upon their recruitment to inflammatory foci, eosinophils become activated in response to a number of local stimuli in the affected tissue. Activated eosinophils release cytotoxic granule proteins, lipid mediators, and products of respiratory burst. Factors thought to be important in recruiting eosinophils into allergen-challenged tissues include cytokines and chemokines [interleukin-3 (IL-3), IL-5, granulocyte–macrophage colony-stimulating factor (GM-CSF), eotaxin and regulated upon activation, normal T-cell expressed and secreted (RANTES)]. 1–3

RANTES is a Cys–Cys (CC) chemokine shown to be a potent chemoattractant for memory T cells, eosinophils, basophils, monocytes/macrophages and mast cells. 3 This chemokine is synthesized by a number of tissue and inflammatory cells within mucosal tissues, and is released in association with allergen challenge in subjects with atopic disease. Elevated levels of RANTES have been found in bronchoalveolar lavage (BAL) fluids from asthmatics within 4 hr of challenge 4 and in nasal lavage fluids from patients with allergic rhinitis following specific allergen challenge. 5 The increase in RANTES mRNA expression and immunoreactivity at these sites strongly correlated with tissue eosinophil numbers. Thus, RANTES has been implicated in the pathogenesis of allergic-type reactions with the potential to contribute to pathological changes observed in allergic inflammation.

Eosinophils have been shown to express RANTES mRNA and its translated protein product, and to release bioactive RANTES in response to physiological stimuli. 6–8 Serum-coated beads (which stimulate receptors for C3b/iC3b; CR1 and CR3, respectively) and interferon-γ (IFN-γ) were found to up-regulate RANTES mRNA and protein expression in eosinophils. 6 We have recently observed that RANTES is stored in at least two intracellular sites in eosinophils, the crystalloid granule and a small, rapidly mobilizable secretory pool, both of which were sensitive to stimulation by IFN-γ. 8 Moreover, the effects of IFN-γ occurred within a shorter period of time than previously observed, with intracellular mobilization of RANTES detectable within 10 min and maximal RANTES release occurring within 60–120 min of incubation. Neither IL-3 nor IL-5 was able to induce significant RANTES release in these experiments. 8 Interestingly, intracellular RANTES immunoreactivity was replenished after 16 hr of IFN-γ stimulation. 8 Since such a rapid change in the distribution of RANTES immunoreactivity in the eosinophil was evident in response to IFN-γ, which was apparently followed by renewed chemokine synthesis and storage, we were interested in investigating the possible regulation of RANTES transcript as a model for potential replenishment processes during cell activation.

We proposed that the expression of RANTES transcripts is differentially regulated in eosinophils, and is dependent on the specific eosinophil-active stimulus. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we provide evidence that RANTES mRNA expression is sensitive to regulation by IFN-γ but not other agonists, including eosinophil-specific cytokines. Complementary to our previous observation of IFN-γ-induced RANTES release in eosinophils, our findings suggest that the eosinophil has the capacity to synthesize new mRNA encoding proteins that have been secreted from intracellular stores as a result of activation.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Materials

Reagents were obtained from the following sources: actinomycin D from Calbiochem Corporation (San Diego, CA); cycloheximide, lipopolysaccharide (LPS), phytohaemagglutinin (PHA) and proteinase K from Sigma (Oakville, ON, Canada); dextran from Fluka Chemie AG (Buchs, Switzerland); DNase from Boehringer Mannheim (Laval, PQ, Canada); Ficoll–Paque from Pharmacia (Uppsala, Sweden); recombinant human IFN-γ (1·3 × 107 U/mg) from Biogen. Res. Corp. (Cambridge, MA); and RPMI-1640 from BioWhittaker (Walkersville, MD). All RT-PCR reagents, including RNase inhibitor, Superscript, and Taq polymerase, were purchased from Gibco BRL Life Technologies (Grand Island, NY). A full-length RANTES cDNA clone was a generous gift from Dr J.F. Elliott, University of Alberta.

Preparation of eosinophils

Samples of peripheral blood (100 ml) were obtained from subjects with mild atopic asthma displaying eosinophilia ≥ 4% and who were not receiving oral corticosteroids. Erythrocytes were removed by dextran sedimentation and the remaining cells were subjected to density centrifugation of Ficoll to obtain a granulocyte pellet. Eosinophils were then purified by negative immunomagnetic selection using the magnetism-activated cell sorting (MACS) system (Miltenyi Biotech, Bergisch-Gladbach, Germany), as previously described. 9–11

In situ RT-PCR

Purified peripheral blood eosinophils were resuspended to a final density of 1 × 106 cells/ml. Three droplets of the cell suspension, containing approximately 40 000 cells/spot, were pipetted onto aminoalkylsilane-coated GENEamp 1000 slides (Perkin-Elmer, Mississauga, ON, Canada) for direct comparison of positive and negative controls with the test sample. Slides were fixed in 4% paraformaldehyde (20 min) before permeabilization with 2 µg/ml proteinase K (10 min). Negative control and test samples were treated overnight at 37° with DNase in 0·1 m sodium acetate, pH 5·0 and 5 m m Mg2SO4. Reverse transcription was performed only in the test sample by adding 50 µl of total volume reaction containing 1X First Strand buffer, 1 m m of mixed dNTPs, 1 µm of specific 3′ primer, 25 U RNase inhibitor, and 500 U Superscript reverse transcriptase. This enzyme was omitted for the both positive and negative controls, while the negative control sample was additionally treated with DNase to prevent detection of either mRNA or genomic DNA sequence. Slides were covered, sealed and incubated for 3 hr at 37° in a GENEamp 1000 machine (Perkin-Elmer). PCR was performed on all samples by adding 18 U Taq polymerase to 50 µl of total reaction volume containing 1X PCR buffer, 4 m m MgCl2, 200 µm of mixed dNTPs (digoxigenin-11 deoxyuridine triphosphate substituted for dUTP) and 0·8 µm each of the specific forward (5′) and reverse (3′) RANTES mRNA primers. PCR was carried out with an initial denaturation step of 95° for 3 min, followed by 35 cycles of 94° for 1 min, 51° for 1 min and 72° for 1 min and 30 seconds, with a final extension step of 7 min at 72°. Digoxigenin-labelled DNA segments were detected by immunohistochemistry using nitroblue tetrazolium/5-bromo-4-3-indolyl phosphate, p-toluidine salt (NBT/BCIP) -conjugated anti-digoxigenin according to the manufacturer’s instructions (Boehringer Mannheim). Total and stained cells were enumerated by counting 500 cells per slide under conventional light microscopy. Staining of cells was carried out using carbol chromotrope-2R, an eosinophil-specific histochemical dye, to co-localize mRNA expression to eosinophils. The 3′ primer sequence for β2-microglobulin was used as the endogenous control.

In vitro stimulation of eosinophils

Serum-coated surfaces were prepared by incubating freshly prepared autologous serum in 1·5-ml Eppendorf tubes for 10 min at 37° and extensively washing the tubes with RPMI-1640 prior to adding cells. The incubation of serum-coated surfaces with eosinophils stimulates their CR1/CR3 receptors. 12,13 Highly purified eosinophils (> 98%, 2 × 106 per sample) were added to serum-coated tubes and incubated at 37° in RPMI-1640 (supplemented with 25 m m HEPES) for 0, 10, 30, 60 and 240 min for in vitro secretion assays, or for 0, 1, 4 and 24 hr for RT-PCR analysis. In assays employing soluble stimuli, 2 × 106 eosinophils were incubated in RPMI-1640 with 5 ng/ml recombinant IFN-γ, 25 ng/ml recombinant human IL-3 (rhIL-3; Genzyme, Cambridge, MA), or 10 ng/ml rhIL-5 (Pharmingen, San Diego, CA) for 1, 4 and 24 hr. Other reagents were also tested on eosinophils for 1 hr, these being 10 ng/ml GM-CSF, 10 ng/ml rhIL-4 (R & D Systems, Minneapolis, MN), 6 n m tryptase (Cortex Biochem, Inc, San Leandro, CA), 10 µl/ml PHA and 5 µg/ml LPS.

RT-PCR

Total RNA was extracted from samples of 2 × 106 highly purified eosinophils (≥ 97%) using RNeasy Mini Kits (QIAGEN, Mississauga, ON) according to the manufacturer’s directions. This procedure routinely yielded approximately 1 µg of total RNA from the sample as determined by absorbance measurement at 260/280 nm. For reverse transcription, 1 µg of total RNA was mixed with 1 µl oligo-dT(12–18-mer) (500 µg/ml), diluted to 11 µl with diethyl pyrocarbonate (DEPC)-treated water, heated to 70° for 10 min, then placed on ice before adding 200 U Superscript reverse transcriptase, 1X First Strand Buffer, and 0·5 m m mixed dNTPs. The mixture was incubated at room temperature for 10 min followed by incubation at 37° for 2 hr. The enzyme was inactivated by heating to 80° for 10 min and the resulting cDNA was stored at −20° until used. The cDNA was amplified by adding 2 µl of sample to 20 µl PCR reaction mix containing 2·5 U of Taq polymerase, 1X PCR buffer, 0·5 m m MgCl2, 25 µm mixed dNTPs, and 0·3 µm each of the appropriate 3′ and 5′ primers. Samples were transferred to a thermal cycler (Perkin-Elmer), and a hot start was performed at 95° for 3 min before samples were subjected to 35 cycles of heating to 95° for 45 seconds, 51° for 1 min, and 72° for 30 seconds. A final extension step was then performed by heating to 72° for 7 min. The amplified products were separated on a 2% agarose gel and subsequently visualized by ethidium bromide staining under ultraviolet illumination. The housekeeping gene, β2-microglobulin, was employed as the positive control for the technique. The primer pair used in the PCR reaction for β2-microglobulin were GCT TAT ATG TCT CGA TCC GAC TTA A (forward primer) and CTC GCG CTA CTC TCT CTT TCT GG (reverse primer) to generate a PCR product of size 335 base pairs (bp). The primer pair for RANTES PCR were TCC CCA TAT TCC TCG GAC (forward primer) and GAT GTA CTC CCG AAC CCA (reverse primer) for a PCR product of size 186 bp. Gel bands were quantified on a gel scanner (Imagemaster DTS, Pharmacia LKB) to allow semi-quantitative analysis of PCR products yielded from these reactions.

Measurement of released mediators

Supernatants and pellets prepared from eosinophils stimulated with appropriate agonists were assayed for eosinophil peroxidase (EPO) and β-hexosaminidase using techniques previously described. 8–11 These samples were also assayed for RANTES using an enzyme-linked immunosorbent assay (ELISA) kit(R & D Systems). The detection limit for the RANTES assay was 31·2 pg/ml. Supernatants were also assayed for leukotriene C4 (LTC4) using a specific ELISA assay obtained from Oxford Biomedical Research, Inc. (Oxford, MI), which had a detection limit of 0·04 ng/ml.

Data analysis

Statistical comparisons were carried out using the Kruskal–Wallis one-way analysis of variance followed by Dunn’s multiple comparison test. Results were considered significant when P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In situ RT-PCR in freshly prepared eosinophils

In order to determine cell-specific expression of mRNA for RANTES in eosinophils, freshly prepared cells were subjected to in situ RT-PCR analysis using RANTES-specific primers as shown in Fig. 1. Message encoding RANTES was found to be constitutively expressed in all eosinophils irrespective of the atopic status of the donor (n = 6). These findings are in agreement with earlier observations using in situ hybridization techniques. 6

image

Figure 1. In situ RT-PCR analysis of RANTES mRNA expression in purified peripheral blood eosinophils. (a) Positive control showing genomic DNA expression of the RANTES gene in eosinophils, detected in cell nuclei using RANTES-specific primers in the absence of a reverse transcription step; (b) negative control in which production of the genomic PCR product was inhibited following preincubation with DNase, while the mRNA sequence was not amplified due to omission of the reverse transcription step; and (c) the test sample, in which DNase was preincubated before reverse transcription to allow detection of RANTES mRNA in the perinuclear and cytoplasmic regions of cells.

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Effects of complement-coated bead stimulation on RANTES release and mRNA expression

We examined the patterns of release of RANTES and other eosinophil-derived mediators following CR1/CR3-mediated stimulation in order to compare this with RANTES transcript expression. Following stimulation of eosinophils in serum-(C3b/iC3b) coated Sephadex beads for 0, 10, 30, 60 and 240 min at 37°, reactions were terminated by centrifugation at 4°. Assays were carried out for RANTES, EPO, β-hexosaminidase and LTC4 in supernatants and pellets ( Fig. 2). Significant EPO and β-hexosaminidase release could not be detected until after at least 240 min of incubation, although RANTES secretion was detectable as early as 60 min. While the changes in stored EPO and RANTES did not reach statistical significance, they showed a decreasing trend during stimulation. RANTES release occurred almost simultaneously with LTC4 release under these conditions ( Fig. 2d).

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Figure 2. Time–course of RANTES release during in vitro stimulation by C3b/iC3b receptor stimulation in comparison with other products of eosinophil activation. Purified eosinophils (2 × 106/sample) were subjected to stimulation by serum-coated Sephadex beads at 37° and the reaction terminated at 0, 10, 30, 60 and 240 min. Eosinophil-derived mediators were measured in supernatants and pellets using appropriate assays for RANTES (a), EPO (b), β-hexosaminidase (c) and LTC4 (d). Open bars indicate released mediators into supernatants, solid bars indicate stored mediators in cell pellets. *P < 0·05, ***P < 0·01. These data are averaged from eosinophils obtained from four to nine atopic subjects.

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We next examined the kinetics of constitutive versus regulated expression of RANTES mRNA of eosinophils following CR1/CR3 stimulation. Cells were incubated with complement-coated beads at 37° for 0, 1, 4 and 24 hr before RNA extraction and RT-PCR analysis. The effects of complement-coated bead stimulation, shown in Fig. 3(a), appeared to be minimal on RANTES mRNA expression, although some up-regulation of message was apparent at 4 hr in this particular example. These data are representative of three separate experiments.

image

Figure 3. Scanned images showing the time–course of RANTES mRNA detection in eosinophils during stimulation by C3b/iC3b and cytokines IFN-γ, IL-3 and IL-5. Eosinophil RNA was extracted from cell pellets following 0, 1, 4 and 24 hr of stimulation at 37° with C3b/iC3b (a), 5 ng/ml IFN-γ (b), 25 ng/ml IL-3 (c) and 10 ng/ml IL-5 (d). RT-PCR analysis was carried out to detect expression of transcripts for RANTES as well as the housekeeping gene, β2-microglobulin.

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Effects of eosinophil-specific cytokines on RANTES mRNA production

To investigate whether eosinophil-active cytokines may exert a regulatory effect on RANTES mRNA expression, eosinophils were incubated with 5 ng/ml IFN-γ, 25 ng/ml IL-3, or 10 ng/ml IL-5, and RANTES mRNA was examined at 0, 1, 4 and 24 hr. Strikingly, IFN-γ was found to induce a significant (P < 0·05) but transient depletion of RANTES mRNA at 1–4 hr which was reversed 24 hr after the start of IFN-γ incubation ( Fig. 3b; Fig. 4b). This result is likely to be IFN-γ-specific, since incubation of eosinophils with IL-3 and IL-5 had no effect on RANTES mRNA expression ( Fig. 3c,d; Fig. 4c,d).

image

Figure 4. Semi-quantitative measurement of RANTES transcripts in stimulated eosinophils. Optical density measurements of RANTES and β2-microglobulin PCR products were plotted as a function of time after 0, 1, 4 and 24 hr stimulation by 5 ng/ml IFN-γ (a), 25 ng/ml IL-3 (b) and 10 ng/ml IL-5 (c). Horizontal bars indicate the mean of three measurements (from three separate atopic donors). *P < 0·05.

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Next we investigated the mechanisms by which IFN-γ induced the observed depletion of RANTES mRNA using the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide. In the first experiment, cells were treated simultaneously with 5 ng/ml IFN-γ and actinomycin D (10−6 m) at time 0. These cells exhibited a more rapid loss of RANTES message detection over time than those incubated with actinomycin D or IFN-γ alone ( Fig. 5a), suggesting that IFN-γ may accelerate the translation of mRNA encoding RANTES. This conclusion was supported by results obtained in the next experiment, in which cycloheximide (10−6 m) was preincubated with cells for 1 hr prior to addition of5 ng/ml IFN-γ, and was found to reverse completely the effects of IFN-γ on RANTES mRNA depletion ( Fig. 5b). The apparently slower rate of down-regulation of RANTES mRNA (shown in Fig. 5a) in response to IFN-γ in this example is likely to be due to donor variation. Taken together, these findings indicate that IFN-γ accelerated the translation of RANTES mRNA, consistent with its effects on RANTES protein release.

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Figure 5. Analysis of RANTES mRNA expression during IFN-γ stimulation. The transcriptional inhibitor, actinomycin D (Act D) (10−6 m), was added to 2 × 106 purified eosinophils with or without IFN-γ (5 ng/ml), and cells were incubated at 37° for 0, 1, 2 and 4 hr (a). In (b), cycloheximide (CHX) (10−6 m), a translational inhibitor, was preincubated with cells for 1 hr at 37° before the start of the experiment and the time–course was carried out as described in (a). Band intensities were determined by densitometry scanning and values were normalized against those of β2-microglobulin. Results are shown as the percentage of the values obtained at time 0 of the treatment.

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RANTES mRNA expression in eosinophils was also assessed in cells stimulated by IL-4 and GM-CSF. We were unable to detect any effect by these cytokines on RANTES mRNA after 1 hr of stimulation ( Fig. 6), although in this particular example, GM-CSF appeared to induce up-regulation of RANTES transcript expression over that of control unstimulated cells. Cell viability did not diminish below 95% using the trypan blue exclusion method after 4 hr of stimulation by any of the cytokines used in this study.

image

Figure 6. Effects of IL-4, GM-CSF, tryptase, PHA and LPS on eosinophil RANTES mRNA expression. RT-PCR analysis of RANTES mRNA was carried out on eosinophil pellets following 1 hr stimulation at 37° with 10 ng/ml IL-4, 10 ng/ml GM-CSF, 6 n m human lung mast cell tryptase, 10 µl/ml PHA, or 5 µg/ml LPS. Pos. control indicates amplified plasmid sequence encoding human RANTES.

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Effects of other agonists on eosinophil RANTES mRNA synthesis

Stimulation of cells by human mast cell tryptase (6 n m) and LPS (5 µg/ml) also failed to induce changes in the apparent expression of RANTES mRNA ( Fig. 6). Tryptase, known to stimulate the release of granule proteins EPO, eosinophil cationic protein (ECP) and β-hexosaminidase from eosinophils, 14 induced moderate (but not significant) RANTES release into supernatants of stimulated cells after 1 hr of stimulation at 37° (approximately 60 pg/ml from 2 × 106 eosinophils; data not shown). 15

The size of the PCR product detected in eosinophils using RANTES-specific primers was identical to that generated by the positive control in these experiments (the full coding sequence of human RANTES, amplified from bacterial plasmid) ( Fig. 6).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this report, we describe a novel finding in which human peripheral blood eosinophil RANTES mRNA expression was found to be transiently down-regulated by the T helper type 1 (Th1) cytokine IFN-γ. The eosinophil-active cytokines, IL-3 and IL-5, had little or no effect on the regulation of RANTES transcript levels, although GM-CSF may have induced some up-regulation of the levels of RANTES mRNA within these cells. Moreover, other eosinophil-specific agonists tested in this study, including serum-coated particles (C3b/iC3b), IL-4, PHA, LPS and human lung mast cell tryptase, did not appear to induce detectable modulation of RANTES mRNA expression.

The effect of IFN-γ on RANTES mRNA expression in eosinophils is compelling, since it suggests that IFN-γ is likely to be a mediator of pro-inflammatory reactions in this important cell. IFN-γ-induced depletion of RANTES mRNA was very rapid, occurring within 1–4 hr of incubation with eosinophils in vitro. Stimulation by IFN-γ has previously been reported to induce up-regulation of RANTES mRNA in eosinophils, but this was observed only after 16 hr of stimulation. 6 The timeframe in which IFN-γ induced the depletion of detectable RANTES mRNA (between 1 and 4 hr) coincides with that of maximal RANTES mobilization and release from intracellular stores in eosinophils (between 1 and 2 hr). 8 The findings in this study suggest that IFN-γ-induced down-regulation of RANTES message is likely to be due to depletion of intracellular levels of RANTES mRNA. Evidence for this was provided by the use of actinomycin D (a transcriptional inhibitor), which accelerated the loss of message in response to IFN-γ, and also cycloheximide (a translational inhibitor), which completely reversed the effects of IFN-γ. The effects of these two inhibitors indicate that IFN-γ may act on eosinophils by accelerating the rate of translation of the available pool of RANTES mRNA beyond that of gene transcription for this chemokine. Consequently, RANTES transcript was depleted to undetectably low levels. A more detailed molecular biological analysis into the mechanisms underlying the acceleration of RANTES mRNA translation in response to IFN-γ is warranted, but remains outside the scope of this study.

The increased rate of RANTES mRNA translation is likely to be accompanied by elevated RANTES protein product release, along with the release of preformed RANTES from granule stores. Interestingly, eosinophil RANTES mRNA was again detectable after 24 hr of stimulation, suggesting that RANTES mRNA was replenished during prolonged IFN-γ incubation. The latter finding correlates with the observation that RANTES immunoreactivity was up-regulated in eosinophils after 16 hr of IFN-γ stimulation. 8

Our findings are supported by previous studies showing that IFN-γ can activate eosinophils by promoting cytotoxicity, 16 inducing and up-regulating surface CD16 and CD69 expression, 17,18 and prolonging the survival of eosinophils. 16 IFN-γ also induces both the up-regulation and release of a number of cytokines from eosinophils, including IL-3, 19 IL-6, 9 IL-12 20 and GM-CSF. 21 These events are likely to be mediated through functional IFN-γ receptors recently described in eosinophils. 22

The effects of C3b/iC3b stimulation on RANTES mRNA expression was also studied since earlier studies showed that RANTES release occurred from eosinophils stimulated via CR1/CR3 receptors, in vitro. 6 Our data confirm that the release of RANTES occurs during serum-coated bead stimulation, which was found to precede that of the granule-stored proteins EPO and β-hexosaminidase. The secretion of RANTES occurred almost concurrently with that of LTC4 release from stimulated cells. Eosinophils have previously been shown to release LTC4 very rapidly following C3b/iC3b-dependent stimulation. 23 However, no significant effects could be observed by incubation of eosinophils with C3b/iC3b on the expression of RANTES mRNA in eosinophils for up to 24 hr. These data suggest that complement stimulation affects neither the transcription nor the translation of RANTES gene product to a significant degree in these cells.

Eosinophils are known to be activated in response to the cytokines IL-3, IL-4, IL-5 and GM-CSF by releasing a battery of mediators, including granule proteins and reactive oxygen species. 24,25 Moreover, all of these cytokines are able to modulate gene expression in eosinophils. 26–28 We therefore investigated the effects of these cytokines on RANTES mRNA expression in comparison with IFN-γ. Preliminary studies have indicated that IL-3, IL-5 and GM-CSF were unable to induce RANTES release from eosinophils. 8 Similarly, we were unable to detect a regulatory effect by any of these cytokines on the expression of RANTES mRNA in eosinophils in the present report. However, we are unable to rule out the possibility that these cytokines may influence RANTES chemokine expression in eosinophils under different experimental conditions than those tested here.

In other tissues, IFN-γ is known to up-regulate RANTES mRNA and induce the release of RANTES protein product in many cell types associated with human atopy, including endothelial cells, 29 nasal fibroblasts 30,31 and mononuclear phagocytes. 32 However, none of these studies examined the effects of IFN-γ on RANTES mRNA and protein release from these cells prior to 12–72 hr of incubation. In addition, the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) has been found to act synergistically with IFN-γ to augment the synthesis of RANTES mRNA in these tissues, in addition to airway smooth muscle cells 33 and a bronchial epithelial cell line. 34

Although it is classified as a Th1-type cytokine based on murine T-cell clone studies, IFN-γ has been shown to be involved in Th2-type reactions in human allergic inflammation and asthma. This adds further fuel to the continued controversy around the putative in vivo existence of a dichotomy of Th1 and Th2 responses. For example, elevated levels of IFN-γ have been detected in the sera of patients with acute severe asthma exhibiting pronounced tissue and lung eosinophilia. 35,36 Other studies have shown elevated levels of IFN-γ in the BAL fluids obtained from mild asthmatics. 37 Moreover, a positive correlation has recently been observed between tissue eosinophilia and IFN-γ mRNA-positive cells in nasal mucosa biopsies in early phase responses (between 1 and 24 hr) following a single allergen challenge in atopic subjects. 38 The majority of T cells infiltrating the airway lumen of asthmatics have been shown to produce IL-2 and IFN-γ, with only a small fraction of these producing IL-4·39 Even in murine studies, the role of IFN-γ in models of allergic and asthmatic inflammation is not clearcut. Using lymphocyte-deficient, ovalbumin-treated severe combined immunodeficiency (SCID) mice, investigators recently demonstrated that the adoptive transfer of IFN-γ-producing Th1 cells into these animals failed to ablate the Th2 cell-induced airway hyperresponsiveness in response to ovalbumin challenge. Instead, the transferred Th1 cells themselves evoked intense airway inflammation. 40 Taken together, these studies suggest that IFN-γ may play a substantially different role in allergic inflammation and asthma than previously appreciated. 41

RANTES is a potent eosinophilotactic chemokine which is up-regulated during allergic and asthmatic inflammation. 4,5,38 It is also synthesized, stored and released by eosinophils purified from the peripheral blood of atopic asthmatics. 6–8 Eosinophil-derived RANTES is bioactive, as shown by its ability to induce chemotaxis in human lymphocytes 7 and human eosinophils. 6 The physiological role of RANTES in inflammation is likely to be important at the level of the local tissue microenvironment rather than systemically. It is also among the first few cytokines up-regulated and/or released following allergen challenge in nasal mucosa 5,42,43 and in BAL fluid of asthmatic individuals. 4 Thus, RANTES is likely to play a prominent role in allergic inflammation, particularly at early stages of the response to allergen.

In conclusion, the eosinophil appears to exhibit a capacity to replenish proteins that have been secreted from intracellular stores as a result of activation. Thus, in this manner, the eosinophil may be able to continue to elaborate mediators and prolong its own survival in tissues. A prominent feature of allergic eosinophilic inflammation is the persistence of activated tissue eosinophils, where otherwise they would undergo apoptosis during the resolution of an inflammatory response. Thus, our findings may shed new light on the mechanisms underlying the persistence of eosinophilic inflammation in allergy and asthma.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors wish to thank Ms Stacey C. Hagen and Mr Ben Bablitz for their assistance with the technical aspects of the study. This work was supported by the Medical Research Council, Canada; Alberta Heritage Foundation for Medical Research, Canada; and by CONACYT Grant no. 3256P-M9608 and Coordination of Biomedical Research, IMSS, Mexico City, Mexico. J.R.V. is the recipient of a fellowship from CONACYT and IMSS; P.L. is a Parker B. Francis Fellow in Pulmonary Research; R.M. is an Alberta Heritage Senior Medical Scholar.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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