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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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To define the effects of immunization and exposure to allergen on the eosinophil lineage, we studied blood and bone-marrow eosinophil numbers, serum interleukin (IL)-5 levels, and eosinophil progenitor and precursor responses to IL-3 and IL-5 in ovalbumin-immunized BALB/c mice after intranasal challenge. Increased blood eosinophilia was found in immune relative to nonimmune mice, but the differences between challenged and unchallenged immune animals were not significant. In contrast, significantly increased circulating levels of IL-5 and numbers of bone-marrow eosinophils were found in sensitized animals exposed to allergen, relative to unchallenged, sensitized controls. An allergen-induced increase in IL-3-sensitive progenitors yielding eosinophil-bearing colonies was also found at 2 h after challenge. Furthermore, an eosinophil differentiation assay showed a marked increase in the magnitude of the responses to IL-5 and IL-3 over a 7-day period in bone-marrow cells of sensitized animals, which was detectable at 24 h after allergen challenge, but not at 2 h and not in unchallenged controls. Modulation of the responses of bone-marrow cells to IL-5 is induced by a circulating factor present in challenged immune animals, as shown by in vivo plasma transfer, but is at best only partly blocked by in vivo treatment with the anti-IL-5 antibody TRFK-5. These data indicate that allergen challenge in the airways leads to rapid long-term modifications in bone-marrow eosinophil progenitors and precursors, and that increased responses to eosinopoietins in bone marrow depend on the release, between 2 h and 24 h after challenge, of a circulating factor distinct from IL-5.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Eosinophils are thought to play a central role in asthma (1), and to contribute to tissue damage through the effect of their secretion products on bronchial epithelium (2, 3) and their ability to secrete a wide array of cytokines (4). T-cell-derived eosinopoietins, such as interleukin (IL)-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5, 6), are thought to control the eosinophilia of asthmatic lung, because activated T-cell and eosinophil numbers in bronchoalveolar lavage fluid (BALF) correlate with the severity of asthma (7) and because anti-IL-5 antibodies suppress pulmonary eosinophilia and hyperreactivity in a number of models (8). Eosinophils in asthmatic airways may be derived from circulating progenitors recruited by inflammatory signals originating in the lung (12), or from bone-marrow cells released after allergen exposure (9). Several reports have described the general effect of allergen exposure on bone-marrow progenitors (12, 13), but few studies have analyzed allergen-induced changes in the bone-marrow eosinophil lineage, and even those did not distinguish between the effects of immunization and of allergen challenge in sensitized animals (9). On the other hand, the issue of whether the response of bone-marrow cells to the eosinopoietins is altered by immunization and/or allergen exposure has not been settled.
It has recently been shown that intranasal challenge of ovalbumin-sensitized BALB/c mice led to increased eosinophil numbers in these animals' BALF, submucosa, and bronchi (11). In the high IgE-producing BP2 mouse strain, development of eosinophil infiltration of the bronchial epithelium and of bronchial hyperreactivity after repeated challenge of sensitized animals was shown to depend on IL-5 (11). Since bone-marrow eosinophil progenitors and precursors are the primary targets of the eosinopoietins, we analyzed the effect of allergen challenge on the responses of bone-marrow cells to IL-5 and IL-3. We report here that allergen challenge of sensitized mice triggers rapid changes in the bone-marrow eosinophil lineage, including a large increase in the responses to eosinopoietins, and provide evidence for the involvement in this modulation of circulating mediators produced after challenge.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Source of Materials
Ovalbumin (5× crystallized) was obtained from ICN Biomedicals, Inc. (Costa Mesa, CA), Al(OH)3 from Merck (Darmstadt, Germany), heat-inactivated fetal calf serum (FCS) from Boehringer Mannheim (Meylan, France), culture media and supplements from Gibco (Life Technologies, Eragny, France), rmIL-3, rmIL-5 and rmGM-CSF from Immunogenex (Los Angeles, CA), Noble agar from Difco (Detroit, MI), Diff-Quik stain from Baxter Dade AG (Dudingen, Switzerland), and Harris' hematoxylin from Réactifs RAL (Paris, France). Monoclonal antibodies (mAbs) TRFK4 and TRFK5 (a gift from Dr. P. Minoprio, Institut Pasteur, Paris, France) and GL113 (a gift from by Dr. H. Savelkoul, Erasmus University, Rotterdam, the Netherlands) were purified from ascitic fluid.
Immunization Procedures and Experimental Design
Animals and immunization procedures. Male and female BALB/c mice (Elevage Janvier, Genest-Saint-Isle, France) were immunized with two subcutaneous 0.4-ml injections of 100 µg ovalbumin mixed with 4 mg/ml Al(OH)3 in 9% NaCl, given 7 days apart (11). One week after the second injection, mice were anesthetized with ether, challenged in both nostrils with 10 µg ovalbumin in 50 µl 0.9% NaCl, and killed at the indicated intervals (Figures 1-3). Experimental groups were as follows: (1) ovo-ovo: immunized and challenged with ovalbumin; (2) ovo-saline: immunized with ovalbumin, challenged with saline alone; (3) Al(OH)3- ovo: sham-immunized with Al(OH)3 alone, challenged with ovalbumin; (4) Al(OH)3-saline: sham-immunized, challenged with saline.
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Blood, Serum, and Plasma Studies
Eosinophil counts were performed at the indicated intervals (Figure 1) on smears of tail blood stained with the Diff-Quik method. Serum IL-5 levels were determined at the same intervals with an enzyme-linked immunosorbent assay (ELISA), using mAbs TRFK-4 for capture and TRFK-5 for development (15). Heparinized plasma was collected from six ovo-ovo and six ovo-saline animals, and 0.5 ml of pooled, unconcentrated plasma of either origin was injected through the tail vein into each of three immune and three nonimmune recipients.
Bone-marrow-cell Studies
Bone-marrow cell harvest. Bone-marrow cells from two mouse femurs were collected in RPMI 1640 medium with 10% FCS, washed, and counted. EPO+ cells were enumerated in cytocentrifuge (Hettich Universal, Tuttlingen, Germany) smears. Progenitor assays. For progenitor quantification (16, 17), semisolid cultures were established in 35-mm culture dishes, following a double-agar-layer procedure (each layer consisting of 0.36% agar in Iscove's modified Dulbecco's medium plus 1 ml of 20% FCS, with or without recombinant cytokines, plus 2 × 105 cells in the top layer). A colony was defined as an ensemble of more than 50 cells, derived from a progenitor, as opposed to a cluster, which had less than 50 cells (18). Colonies were scored at day 7 under the inverted microscope at low magnification, and the frequency of colonies bearing eosinophils was determined on agar layers that were dried (50°C), mounted on microscope slides, stained for EPO as described below, counterstained with Harris' hematoxylin, and scored under high magnification. IL-5 did not support true colony formation, but did support cluster formation. Precursor assays. Liquid bone-marrow cultures (106 cells in a 1-ml volume, in a 24-well cluster) were seeded in RPMI 1640 medium with 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin (Gibco Europe) and were maintained at 37°C under 5% CO2/95% air for 7 days. The frequency of EPO+ cells was determined in cytocentrifuge smears. The concentrations of IL-5 used in this study were defined by the ability of this cytokine to stimulate eosinophil differentiation in liquid culture, based on the criteria of Strath and colleagues (14). Unless otherwise indicated, IL-5 was used at 2, 4, and 6 ng/ml final concentrations. Although less effective than IL-5, IL-3 was active in liquid culture at 0.1 ng/ml and 1 ng/ml (see RESULTS). Staining for eosinophil peroxidase. Staining followed the protocol of Ten and colleagues (20). The cytochemical pattern of EPO+ cells in stained bone-marrow preparations was identical to that reported by Horton and associates (21). Comparable results could be obtained on cytocentrifuge smears with conventional eosin stains. However, visualization and counting were much easier after EPO staining, especially for immature forms such as early precursors, because EPO is present in both mature and immature forms even when eosinophilic granules are not prominent. Confirming earlier reports (14, 21, 22), bone-marrow neutrophils were not stained. Discontinuous density separation of bone-marrow cells. Gradient separation of bone-marrow cells was done as previously described (23). Isotonic Percoll (100%; Sigma Chemical Co., St. Louis, MO), prepared in phosphate-buffered saline (PBS), was diluted to 75%, 60%, and 40% (2 ml, 3 ml, and 2 ml per gradient, respectively). Bone-marrow cells (1 femur equivalent/two gradients) were separated at 100 × g for 20 min at 20°C. Cells at the three interfaces (Layers I to III from top to bottom) were collected, washed, and counted. To confirm that the gradient efficiently separated immature from mature cells, the presence of progenitors in each layer was assessed by colony formation. In the presence of 1 ng/ml IL-3, comparable numbers of colonies were formed by 2 × 105 unseparated bone-marrow cells or by 105 Layer II cells. No colonies were observed upon culture of Layer III cells. Layer I contained mostly debris. On the basis of the 2-fold enrichment for immature cells, liquid cultures of density-gradient-separated cells were established with 5 × 105 Layer II cells, in the presence of IL-5, for 7 days. For liquid culture, Layer III cells were seeded at 106/ml.Statistical Analysis
The data were initially subjected to factorial analysis of
variance (ANOVA) to determine how the independent
variables, or factors (group, time after challenge, presence
of antibody, nature of donor), affected the dependent variables (blood and marrow eosinophilia, IL-5 levels). For
each of the kinetic studies shown in Figures 1-3,
the data were further submitted to unifactorial ANOVA, in
which the independent variable was the time after challenge, to determine how this factor affected the dependent
variable within each group. Subsequently, for multiple between-group comparisons of means, the Tukey (honestly
significant differences) (HSD) correction was used. P 
0.05 was considered significant and P 0.01 was considered
highly significant. Because the Systat for Windows version
4 software was used for the data analysis, missing values would be generated only by the elimination of outliers or
by transformation errors, and in none of the cases presented
in the RESULTS section did statistical significance depend
on either of these situations.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of Sensitization and Challenge on Blood Eosinophil Numbers
All four groups of sensitized and nonsensitized mice were analyzed for peripheral blood eosinophil numbers at various intervals after exposure to ovalbumin or saline. As shown in Figure 1, immunization led to increased numbers of blood eosinophils in immunized and challenged (ovo- ovo) animals relative to both nonsensitized control groups, with significant differences at the time points of 6 h and 24 h. However, despite a trend toward larger mean values in the ovo-ovo animals, the differences between challenged and unchallenged immune animals were not significant.
Effect of Sensitization and Challenge on Blood IL-5 Levels
Serum levels of IL-5 in all four groups of mice were also determined after exposure to allergen or saline. As shown in Figure 2, mean levels of circulating IL-5 were generally increased in the ovo-ovo animals relative to the other groups, especially at 24 h after allergen exposure. IL-5 levels in ovo-ovo mice were significantly increased relative to both groups of nonsensitized controls at 24 h. Furthermore, the difference between ovo-ovo and ovo-saline animals was also significant at 24 h. Hence, in contrast to peripheral blood eosinophilia, serum IL-5 levels were significantly increased by allergen exposure of immune animals relative to nonchallenged controls.
Effect of Sensitization and Challenge on Eosinophil Numbers in Bone Marrow
Eosinophil numbers in bone-marrow cell populations were also analyzed after allergen challenge in all four groups of sensitized and nonsensitized mice. As shown in Figure 3, immunization itself resulted in an increase in marrow-cell populations relative to nonimmune animals: ovo-saline animals had significantly increased numbers of eosinophils relative to alum-saline and alum-ovo mice at 2 h after challenge. However, a very significant increase in eosinophilia was also detected in bone marrow of ovo-ovo mice relative to nonchallenged immune controls at 2 h, 6 h, and 24 h after allergen exposure. Hence, in contrast to blood eosinophilia, bone-marrow eosinophilia showed a selective effect of allergen provocation. Further, in contrast to increased serum IL-5 (see the preceding discussion), this effect was detectable as soon as 2 h after challenge, and lasted up to 24 h.
Effect of Allergen Challenge on Eosinophil Progenitors
The ability of IL-3 to stimulate eosinophil progenitors was
initially evaluated with the bone marrow of ovo-ovo animals. Cultures established in the presence of 1 ng/ml IL-3
contained 64 ± 3.1 colonies/2 × 105 cells (mean ± SEM,
n = 4), allowing easy visualization of individual colonies
after EPO staining and counterstaining. In contrast, IL-3
at 0.1 ng/ml supported only 5 ± 0.78 colonies/2 × 105
bone-marrow cells (mean ± SEM, n = 7), and the concentration of 1 ng/ml was used in the subsequent experiments.
EPO-stained colonies corresponded to the morphologic
and cytochemical patterns described by Zucker-Franklin
(19), and consisted of both pure eosinophil colonies (derived from eosinophil colony-forming units [CFU-EOS]
[16, 17]) and mixed colonies containing eosinophils, granulocytes, and monocytes/macrophages (derived from granulocyte/macrophage/eosinophil colony-forming units [CFU-G/M/EOS] [17]). As shown in Figure 4A, pure eosinophil
colonies were small and compact, in agreement with the
findings of Chervenick and colleagues (16) and Zucker-Franklin (19), whereas mixed colonies were larger and less
compact (Figure 4B). As shown in Figure 4C, pure colonies contained only eosinophils. As shown in Figure 4D,
eosinophils in a mixed colony were a significant proportion of all cells in the colony, and EPO
granulocytes and monocytes, present in the same colony, were revealed by
nuclear morphology after counterstaining.
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When the ability of bone-marrow cells to form eosinophil-bearing (both pure and mixed) colonies in semisolid media was examined, a rapid and selective effect of allergen challenge was also detected. As shown in Table 1, the frequency of colonies containing EPO+ cells was significantly increased in ovo-ovo animals relative to ovo-saline controls as early as 2 h after allergen exposure when cells were cultured for 7 days in the presence of IL-3. In contrast, as shown in Table 1, no differences between ovo-ovo and ovo-saline immune animals were observed in the representation of the different subtypes of myeloid colonies that were devoid of eosinophils (G, M, and GM).
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Effect of Allergen Challenge on the Response of Bone-marrow Cells to IL-5
The ability of IL-5 to stimulate eosinophil precursors was initially evaluated in ovo-ovo animals. As shown in Table 2, cultures established in the presence of 0.6 to 6 ng/ml IL-5, from unseparated bone marrow, showed increased numbers of EPO+ cells relative to uncultured bone marrow, which increased with increasing IL-5 concentrations and plateaued at concentrations of 2 to 6 ng/ml. The increase in numbers of EPO+ cells in stained cytocentrifuge smears corresponded to an actual 4- to 5-fold increase in the absolute number of EPO+ cells in the culture relative to their numbers in the original bone-marrow inoculum, which contained (0.8 ± 0.1) × 105 EPO+ cells/ml (mean ± SEM, n = 25). As shown in Figure 5A, IL-5-stimulated cultures at day 7 presented a majority of EPO+ cells displaying the morphologic characteristics of precursors (large cell size and large, unsegmented nucleus with loose chromatin), along with mature eosinophils (recognizable by a small cell size and donut-shaped nucleus with compact chromatin). The same cultures contained neutrophils, macrophages, and other hemopoietic cell types. In contrast, cultures kept for 7 days in medium alone contained few or no EPO+ cells, and most cells detectable with counterstaining presented morphologic signs of degeneration (Figure 5B).
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Since the EPO+ cells in uncultured bone marrow included some mature eosinophils, cell-separation studies were done to evaluate the possible contribution of mature eosinophil survival to the increased EPO+ cell numbers at day 7. Bone marrow was separated on the basis of density differences between immature and mature cells (see MATERIALS AND METHODS). The intermediate-density Layer II was enriched in blast forms and contained large numbers of progenitors, as detected in colony-formation assays, but contained no mature eosinophils, as determined from the nuclear morphology of EPO+ cells in cytocentrifuge smears. The most dense layer, Layer III, contained mature eosinophils but no detectable progenitors. As shown in Table 2, Layer-II cells did respond vigorously to the concentrations of IL-5 that induced plateau responses in unseparated bone marrow (2 to 6 ng/ml). However, unlike unseparated bone marrow, cultures of Layer-II cells established in the presence of 0.2 to 0.6 ng/ml IL-5 did not present EPO+ cells. Such cultures were paucicellular and presented widespread degeneration, showing that Layer-II cells were more dependent on high IL-5 concentrations for survival and in vitro eosinopoiesis than was unseparated bone marrow. On the other hand, cultures of Layer-III cells established in the presence of IL-5 at concentrations of 0.2 to 6 ng/ml did not present EPO+ cells and were paucicellular, with debris and degenerating cells.
When eosinophil differentiation over a 7-day period of liquid culture in the presence of IL-5 was compared for all groups of animals, further evidence was obtained for selective effects of allergen challenge on bone-marrow cells. As shown in Figure 6B, bone-marrow cells from ovo-ovo animals had greatly increased responsiveness to IL-5 at 24 h after challenge. This increase was highly significant with regard to all control groups. However, in contrast to the responses of progenitors to IL-3 (see the preceding discussion), increased responses of eosinophil precursors to IL-5 could not be demonstrated in bone-marrow cells that had been harvested only 2 h after allergen exposure (Figure 6A).
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Effect of Allergen Challenge on Bone-marrow-cell Responses to IL-3 in Liquid Culture
An increased response in an assay for eosinophil differentiation factors (14) was not limited to the effects of IL-5, since similar observations were made when the response to IL-3 in liquid culture was compared for all groups of animals. Figure 7 shows that both ovo-ovo and ovo-saline mice had significantly increased responses to IL-3 relative to the alum-ovo controls when bone-marrow cells were harvested 2 h after allergen challenge (Figure 7A). Under these conditions, however, the difference between the responses of ovo-ovo and ovo-saline animals was not significant. In contrast, when bone-marrow cells were harvested 24 h after challenge, the response of cells from ovo-ovo mice to 1 ng/ml IL-3 was significantly greater than that of cells from ovo-saline controls (Figure 7B). This was not observed when cells harvested at 24 h were cultivated in the presence of 0.1 ng/ml IL-3, indicating that an optimal IL-3 concentration is needed for this selective effect of allergen exposure.
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Evidence for a Circulating Factor Involved in Modulating Bone-marrow-cell Responses to IL-5
The possibility that a circulating factor, produced in response to allergen challenge, was responsible for the increase in magnitude of the response of bone-marrow cells to IL-5 was investigated in transfer experiments. Plasma was collected from ovo-ovo and ovo-saline animals at 24 h after ovalbumin or saline challenge, and its ability to modulate the response to IL-5 in bone-marrow cells was assayed by transfer to immune and nonimmune recipient mice. To evaluate the selectivity of the effect for IL-5, responses of the same cells to GM-CSF were evaluated in parallel. As shown in Table 3, bone-marrow cells from both immune and nonimmune recipients of plasma from ovo-ovo mice showed a significantly increased response to IL-5 relative to bone-marrow cells from immune and nonimmune recipients of plasma from ovo-saline mice. This pattern was not observed for responses to GM-CSF, although there was a somewhat greater response to the latter cytokine in immune recipients of plasma from ovo-ovo mice. Hence, the effect of challenge on the responses of bone-marrow eosinophil precursors to IL-5 can be duplicated by a plasma factor, or factors, present 24 h after challenge.
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Effect of In Vivo Anti-IL-5 Antibody Treatment on Responses to Eosinopoietic Cytokines
The possibility that the circulating factor responsible for modulating the response of bone-marrow cells to IL-5 was IL-5 itself was investigated in a protocol in which immune mice were treated 1 h before challenge with the TRFK-5 anti-IL-5 neutralizing mAb or with the isotype-matched control antibody GL113. Figure 8 shows that the responses to IL-5 in liquid culture of bone marrow from mice injected with anti-IL-5 were significantly weaker at all IL-5 concentrations tested than were the responses of bone marrow from mice injected with the control antibody. This inhibition, however, was partly overcome by culturing bone-marrow cells in the presence of higher concentrations of IL-5. Besides, even at the best inhibition achieved, the response was more intense than at 2 h after challenge (Figure 6A). These results indicate that anti-IL-5 antibody did not completely prevent the effect of allergen challenge on the magnitude of the response of bone-marrow cells to IL-5. This cannot be attributed to the use of insufficient amounts of anti-IL-5 antibody, since its in vivo effectiveness was confirmed in parallel experiments, in which it significantly reduced bone-marrow eosinophilia and virtually eliminated IL-5 from the circulation (Table 4).
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In vivo treatment with anti-IL-5 antibody also modulated the responses to GM-CSF. As shown in Table 5, anti-IL-5 antibody did not affect the total numbers of myeloid colonies formed in the presence of GM-CSF, although some decrease in the frequency of eosinophil-containing colonies (pure and mixed) could be observed. Unexpectedly, however, the antibody did significantly inhibit the differentiation of eosinophils in liquid culture in response to GM-CSF (Table 5). On the one hand, these data provide further evidence for the in vivo effectiveness of the anti-IL-5 antibody treatment. On the other hand, they suggest the involvement of IL-5 in the in vitro response to GM-CSF in liquid culture.
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Our findings indicate that airway exposure to allergen has profound effects on eosinophil progenitors and precursors in the bone marrow, including a rapid modulation of colony formation and terminal differentiation. In contrast, we could not find an association between allergen exposure and blood eosinophilia under the same conditions, possibly because of rapid turnover of eosinophils coming from bone marrow and exiting to the tissues. Our data suggest that rapid changes in EPO+ cell numbers in the bone marrow more closely reflect the effect of recent allergen exposure than do similar changes in peripheral blood.
Allergen exposure selectively increased EPO+ cell numbers in bone marrow as early as 2 h after challenge, as reported in other strains of mice (27). More significantly, at the same early time point (Table 1), we observed an allergen-specific increase in the ability of eosinophil progenitors to respond to IL-3 by forming eosinophil-containing colonies in agar.
The most striking finding in this study was that of greatly increased bone-marrow responses to IL-5, and to a lesser extent, to IL-3, in an assay for eosinophil differentiation factors induced specifically by allergen exposure. The possibility that increased EPO+ cell numbers, in response to IL-5 or IL-3, resulted from increased survival of mature eosinophils, present in the ovo-ovo bone-marrow inoculum in increased numbers, was directly ruled out on the basis of: (1) the cell-separation studies, which showed that immature cells, depleted of mature eosinophils, strongly responded to IL-5, whereas mature cells, including eosinophils, were unable to do so; (2) the morphology of the EPO+ cells in 7-day cultures (Figure 4A), consisting largely of immature forms rather than mature eosinophils; and (3) the demonstration of a 4- to 5-fold increase in the absolute numbers of EPO+ cells over those in the original ovo-ovo bone-marrow inoculum after 7 days of culture. Overall, our findings are compatible with an allergen-induced increase in the numbers of progenitors and in the responses of precursors, but not with an increased survival of mature eosinophils.
Several studies have analyzed the stimulatory effects of allergen challenge on bone-marrow progenitors (9, 12, 13), but the nature of the target cells and the extent of their ability to terminally differentiate into eosinophils have not been thoroughly defined. Our data point to two sets of targets. The first are the IL-3-responsive progenitors that give rise to pure eosinophil colonies (16, 17, 19) and to colonies containing eosinophils, granulocytes, and monocytes/ macrophages (17), the frequencies of which are increased by about 4-fold as early as 2 h after allergen exposure. The second set of targets are the IL-5-responsive precursors (14), for which a similarly increased response could be detected at 24 h but not at 2 h after challenge. These latter precursors may be identical to the precursors that show smaller responses to IL-3 under the same conditions, since IL-5 and IL-3 have different potencies on the eosinophil lineage (1, 24). Both progenitors and precursors yield terminally differentiated eosinophils.
The data strongly suggest that a critical event, taking place in the immune animal between 2 h and 24 h after contact of allergen with the nasal mucosa, leads to an increased ability of eosinophil progenitors and precursors to respond in vitro to IL-3 and IL-5. This event is most likely the appearance of a circulating factor, since this would mediate a long-term effect on bone marrow from the site of a local inflammatory reaction. We have provided evidence for such a factor through the successful transfer, from challenged to nonchallenged immune animals, of increased responses of the bone marrow to IL-5 by the injection of plasma collected at the appropriate time point (24 h). Since such plasma transfer also induced increased responses to IL-5 in bone-marrow cells from nonimmune mice, the circulating moiety found in challenged but not in nonchallenged mice is sufficient to promote this modulation, regardless of other factors contributed by preexisting immunity. However, our data argue against a role for in vivo-produced interleukins in this allergen-induced modulation of bone-marrow eosinophil precursors, because: (1) bone- marrow cells collected at either 2 or 24 h after challenge were subsequently kept in the presence of IL-5 or IL-3 for as long as 7 days, and it is therefore unlikely that a shorter exposure to in vivo-produced IL-5 or IL-3 accounts for the much weaker responses of cells collected at 2 h; and (2) in vivo treatment with anti-IL-5 antibody only partly inhibited the allergen-induced change. This contrasts with the findings in Nippostrongilus brasiliensis-infected BALB/c mice, in which TRFK-5 antibodies prevent the increase in bone-marrow eosinophil numbers and the generation of IL-5-responsive precursors (26). These discrepancies may reflect differences between the responses to purified protein and parasite antigens, or the involvement of immunomodulatory factors. On the other hand, it is important to define whether this modulation entails the upregulation of receptor number and/or affinity, possibly involving the signaling subunits known to be shared by IL-3 and IL-5 receptors (24).
Inman and colleagues (25) have reported the increased formation of GM colonies in the presence of colony-stimulating factor (CSF), and autologous serum in sensitized dogs, which develop airway hyperresponsiveness in response to inhalation of Ascaris suum antigens. They also described a challenge-induced serum factor, present in responder but not in nonresponder dogs, that upregulates the progenitor response to these factors. Although our findings confirm that challenge modulates bone-marrow eosinophil progenitors through allergen-induced circulating factors, our data differ from those of Inman and colleagues in several important respects, as follows: (1) we found no evidence for modulation of GM colony formation; (2) we further demonstrated a major effect of allergen challenge on IL-5-responsive precursors, pointing to a lineage-specific regulation of eosinopoiesis; (3) production of the plasma factor was dissociated from airway hyperresponsiveness, because ovo-ovo BALB/c mice do not become hyperreactive, even though they present increased eosinophil numbers in BALF, eosinophil infiltration, and eosinophilic vasculitis (11); and (4) the murine plasma factor in our study was equally effective on bone marrow of immune and nonimmune mice, an issue as yet unsettled in the canine model (25).
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Footnotes |
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Address correspondence to: Maria Ignez C. Gaspar Elsas, Departamento de Pesquisa, Instituto Fernandes Figueira, FIOCRUZ, Rio de Janeiro, Av. Rui Barbosa 716, Rio de Janeiro 22250-020, Brazil.
(Received in original form June 25, 1996 and in revised form January 14, 1997).
Acknowledgments: This work was supported by the Institut National de la Santé et de la Recherche Medicale (INSERM), Institut Pasteur, PAPES/ FIOCRUZ, RHAE-UFRJ, CNPq and CAPES-COFECUB. The authors thank Dr. Maria de Fátima S. Costa for statistical advice and calculations.
Abbreviations ELISA, enzyme-linked immunosorbent assay; EPO+, eosinophil peroxidase-positive; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin.
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References |
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Materials & Methods
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Discussion
References
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1.
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