Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities.

Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.     

Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor alpha

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2'-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2'-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R alpha-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.     

IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity

Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.     

Evidence for local eosinophil differentiation within allergic nasal mucosa: inhibition with soluble IL-5 receptor

Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5Ralpha+ cells) and IL-5 mRNA+ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5Ralpha (sIL-5Ralpha), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5Ralpha mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA+ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5Ralpha. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5Ralpha.     

Cutting edge: IL-5 primes Th2 cytokine-producing capacity in eosinophils through a STAT5-dependent mechanism

Both type-2 CD4(+) Th cells (CD4(+)Th2) and type-2 innate effector cells play critical roles in generating type-2 immunity that can either be protective against parasitic infection or cause tissue damage in allergy and asthma. How innate effector cells acquire the capacity to produce Th2 cytokines is not entirely known. We previously showed that IL-4 induced differentiation of Th2 cytokine-producing eosinophils. To determine whether other Th2 cytokines can also induce Th2 cytokine-producing capacity in innate effector cells, we cultured bone marrow progenitor cells in the presence of various Th2 cytokines. IL-5, but not IL-13 or IL-25, primed bone marrow progenitor cells to differentiate into robust IL-4-producing cells. The majority of IL-4-producing cells induced by IL-5 were eosinophils. Importantly, IL-5 completely depended on STAT5 to promote IL-4-producing capacity in eosinophils. Thus, our study demonstrates that IL-5 functions as a potent factor that drives bone marrow progenitor cells into IL-4-producing eosinophils.     

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli

To characterize interleukin (IL)-5-induced eosinophils, we examined the expression of CD44, very late antigen (VLA)-4, and the IL-5 receptor alpha chain, as well as the levels of eosinophil peroxidase and the generation of superoxide. Eosinophils were prepared from IL-5-transgenic mice, then characterized using electron microscopy to determine their responses to stimuli. Whereas CD44 densities remained almost constant, the level of VLA-4 increased in parallel with eosinophil maturation. Although a subset of IL-5-induced eosinophils with high side scatter recovered from bone marrow and rare ones found in blood recognized hyaluronic acid (HA), most did not have this property. Bone marrow eosinophils with high side scatter and lower density contained eosinophil peroxidase, not only in granules, but also in membranous structures for 30% of this population. This population developed HA-binding ability in response to IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, eotaxin, nerve growth factor (NGF), and opsonized zymosan (OZ). Peripheral blood eosinophils acquired HA-binding ability in response to the same stimuli, but their responses were less than those of bone marrow eosinophils with high levels of side scatter. However, splenic eosinophils did not respond to these stimuli. Although peripheral blood eosinophils did not proliferate when stimulated by IL-5, these were the only cells that released eosinophil peroxidase in response to IL-4, MIP-2, MCP-1, eotaxin, NGF, and OZ. With the exception of a subset of bone marrow eosinophils, the ability to acquire HA binding, but not the ability to generate superoxide, correlated with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing cells.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of interleukin 5 receptors on murine eosinophils: a comparison with receptors on B13 cells

The interleukin 5 receptor (IL-5R) on murine eosinophils and a mouse B cell line (B13) was investigated using iodinated murine IL-5 produced in the baculovirus system. Electrophoretic analysis of this recombinant protein identified a range of bands Mr 26,000 to 32,000 resulting from differential glycosylation. The specific activity and binding kinetics of the iodinated IL-5 (125I-IL-5) were essentially identical to unlabeled material. Both high-affinity (Kd approximately 50 pM) and low-affinity (Kd approximately 1 nM) receptor populations were identified on murine eosinophils. Approximately 50 high-affinity receptors and 10,000 low-affinity receptors were present. This was compared with approximately 2,000 high-affinity (Kd approximately 80 pM) and about 8,000 low-affinity (Kd approximately 3 nM) sites on B13 cells. An antibody that inhibits IL-5 binding to, and proliferation of, B13 cells (R52.120) was also shown to inhibit eosinophil proliferation, suggesting that eosinophils and B cells bear the equivalent IL-5 binding proteins.     

Characterization of the human IL-5 receptors on eosinophils

Interleukin 5 (IL-5) receptors on the cell surface of human eosinophils and other hematopoietic cells were characterized using radiolabeled recombinant IL-5. The binding of 35S-labeled murine IL-5 to eosinophils from normal human peripheral blood was rapid and saturable within a 30-min incubation at both 4 and 37 degrees C. The binding of 35S-labeled murine IL-5 to eosinophils was inhibited by an excess of unlabeled murine and human IL-5 or by an anti-murine IL-5 monoclonal antibody (NC17) but not by other human cytokines. Scatchard plot analysis revealed that human eosinophils have a single class of high affinity receptor (Kd 170-330 pM; number of binding sites: 260-380/cell). IL-5 receptors on eosinophils from four patients with eosinophilia displayed similar characteristics. Affinity cross-linking experiments resulted in the identification of human IL-5 receptor on eosinophils with a molecular mass of 55-60 kDa. Among the various cells besides eosinophils and cell lines that we could test, a subline of HL-60 (YY-1 cells) was found to display a significant number of IL-5 receptor. These results suggest that IL-5 may act on limited types of cells in the human system.     

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.     

IL-4 induces differentiation and expansion of Th2 cytokine-producing eosinophils

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.     

Airway allergen exposure stimulates bone marrow eosinophilia partly via IL-9

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2''-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Eosinophils and Megakaryocytes Support the Early Growth of Murine MOPC315 Myeloma Cells in Their Bone Marrow Niches

Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.     

Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.     

The in vivo effects of interleukin 1. I. Bone marrow cells are induced to cycle after administration of interleukin 1

We have previously reported that interleukin 1 (IL-1) administration 20 hr before irradiation protects mice from lethal effects of radiation. The recovery of total nucleated bone marrow cells and of hematopoietic progenitor cells was enhanced in IL-1 treated, as compared to untreated, irradiated mice. This suggested that IL-1 administration may affect the cells in the bone marrow of normal mice. Intraperitoneal administration of recombinant IL-1 resulted in bone marrow cell enlargement and increased cycling of these enlarged cells. In addition, the capacity of bone marrow cells from IL-1 treated mice to proliferate in response to granulocyte macrophage-colony-stimulating factor (GM-CSF) in cell suspension cultures was enhanced. The above effects were not genetically restricted as C57BL/6, B6D2F1, C3H/HeN, and C3H/HeJ mice showed similar responses. A comparative study showed that 100 ng of IL-1 was much more effective in stimulating bone marrow cells by the above criteria than 5 micrograms GM-CSF. Since IL-1, unlike CSF, can not be demonstrated to have a direct in vitro stimulatory effect on bone marrow cells, the aforementioned in vivo effects of IL-1 are presumably mediated by other hematopoietic growth factors. We have previously shown that IL-1 induces the appearance of high titers of CSF in the serum. Consequently hematopoietic growth factors that are generated at local sites following IL-1 administration may mediate the observed cell cycling effect.     

Possible participation of a JAK2 signaling pathway in recombinant rat interleukin-5-induced prolongation of rat eosinophil survival

Recombinant rat interleukin (IL)-5-induced prolongation of rat eosinophil survival in culture was inhibited in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A when examined 96 h after incubation. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that the activation of JAK2 tyrosine kinase and protein synthesis are required for the prolongation of rat eosinophil survival induced by recombinant rat IL-5. STAT5 phosphorylation might also participate in the IL-5-induced survival of rat eosinophils.     

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.     

IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome

This study was undertaken to evaluate the role of IL-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum IL-5 levels that might be essential for eosinophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-IL-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of IL-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce IL-5.