(Chest. 2003;123:345S-348S.)
© 2003
American College of Chest Physicians
Isolation and Characterization of Hemopoietic Cells From Lungs of Allergic Mice*
Maria Ignez C. Gaspar Elsas, MD, PhD;
Elisabeth S. Maximiano, PhD;
Danielle Joseph;
Adriana Bonomo, MD, PhD;
Bernardo Boris Vargaftig, MD, PhD and
Pedro Xavier Elsas, MD, PhD
* From the Departamento Pediatria (Dr. Elsas), Institute Fernandes Figueira, FIOCRUZ, Rio de Janeiro, Brazil; Department of Immunology (Drs. Maximiano, Bonomo, and Elsas), Institute Microbiologia Professor Paulo de Góes, UFRJ, Rio de Janeiro, Brazil; and Unité de Pharmacologie Cellulaire (Ms. Joseph and Dr. Vargaftig), Unité Associée Institut National de la Santé et de la Recherche Médicale-Institut Pasteur U485, Paris, France.
Correspondence to: P. Xavier Elsas, MD, PhD, Associate Professor, Department of Immunology, Instituto de Microbiologia Prof. Paulo de Góes, Universidad Federale do Rio de Janeiro, CCS, Bloco I, 2° andar, sala 066, 21941–590, Rio de Janeiro, Brazil; e-mail: pxelsas{at}yahoo.com.br
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Abstract
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We developed a procedure for the isolation of hemopoietic cells from murine lung. Ovalbumin sensitization and challenge increased the numbers of functionally intact hemopoietic progenitors recovered from digested lung fragments by 80-fold to 120-fold, relative to naive controls. Eosinophil precursors, which are absent in the naive mouse lung, accumulated in the lungs of sensitized/challenged mice. Progenitors in allergic BALB/c mice were recoverable from lung parenchyma, not blood or airways, and were exclusively CD34+. Precursors isolated from allergic lung, unlike those from bone marrow, were inhibited by dexamethasone and were stimulated by prostaglandin D2. This directly demonstrates that sensitized/challenged lungs accumulate hemopoietic progenitors and precursors, distinct from those in bone marrow.
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Introduction
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In humans, the accumulation of CD34+/interleukin (IL)-5 receptor-
messenger RNA+ cells, which presumably include eosinophil progenitors, has been described in the allergen-challenged lung.1
However, direct evidence for the presence of functionally intact hemopoietic progenitors in the lungs has not yet been reported because this involves procedures and assays that are difficult to carry out with human lungs. Here we describe a procedure for the quantitative recovery of fully functional hemopoietic cells, which accumulate in allergen-challenged mice, from murine lungs.
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Materials and Methods
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Animals and Animal Procedures
BALB/c mice were immunized following either of the following protocols: (1) sensitization by two subcutaneous 0.4-mL injections of 100 µg ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 (alum) in 0.9% NaCl (saline solution), at 7-day intervals, followed by intranasal challenge with 10 µg OVA in 50 µL saline solution, 1 week after the second injection2
; and (2) sensitization at day 0 by one intraperitoneal 0.2-mL injection of 10 µg OVA mixed with alum in saline solution, followed by intranasal challenge at 24-h intervals with 20 µg OVA in a 50-µL saline solution from day 10 to day 13. Controls were either naive or sensitized mice that had been challenged with saline solution. Animals were killed 24 h after the last challenge. Mice that were anesthetized with thiopental were exsanguinated from the abdominal vena cava. The heparinized blood was used for hemopoietic cell purification. After opening the thoracic cage, an incision was made in the right atrium, and the remaining blood was washed out of the lungs by pumping sterile saline solution through the left atrium. The effectiveness of this procedure in clearing the pulmonary microcirculation was confirmed by histologic examination of anti-CD34-stained lung sections (Tissue-Tek; Elkhart, IN). The lungs were excised and minced in Iscove modified Dulbecco medium (IMDM) in a Petri dish. BAL fluid, blood, and/or spleen cells, in addition to lung tissue, were harvested from naive or sensitized mice.
Isolation and Quantification of Hemopoietic Cells
Turk solution was used for nucleated cell counts; trypan blue was used for the assessment of viability and staining for eosinophil peroxidase (EPO)3
for the identification of mature and immature eosinophils. Minced lungs were incubated for 20 min at 37°C, with a mixture of 95% air/5% CO2, in 10 mL IMDM containing 24 mg collagenase and 0.125 mg DNAase I, before the addition of fetal calf serum (FCS; 20% final) to stop digestion. Lung cell suspensions were obtained by passing fragments through a cell strainer (Falcon 2360) [100 µm, nylon], washed twice, counted, and separated on discontinuous gradients (Percoll; Amersham Bioscience; Little Chalfont, Buckinghamshire, UK) [75%, 60%, and 40% Percoll in IMDM] for 20 min, at 100g, at 20°C, with 2 x 107 cells per gradient. The low-density mononuclear cells at the 40 to 60% Percoll interface (layer 2) were harvested, washed twice in RPMI medium/FCS, counted, and used for EPO staining and hemopoietic assays. Where indicated, they were stained with rat antimouse CD34 antibody and goat anti-rat IgG fluorescein isothiocyanate conjugate, and were analyzed by flow cytometry (FACScalibur; Becton Dickinson Biosciences; Franklin Lakes, NJ) or were further incubated with fluorescein isothiocyanate (MicroBeads; Miltenyi Biotech GmbH; Cologne, Germany) for 15 min and separated (MidiMACS column; Miltenyi Biotec). For bone-marrow, BAL fluid, blood, and spleen cells, triplicate 1-mL semi-solid cultures were plated in methylcellulose in 35-mm culture dishes from 2 x 105 cells, in IMDM with 20% FCS, granulocyte-macrophage colony-stimulating factor (GM-CSF) [2 ng/mL], IL-3 (2 ng/mL), and IL-5 (1 ng/mL). Colonies were scored at days 7 and 10. Scoring after day 7 did not result in better colony detection, but colony morphology was less preserved. For comparison with lung-derived colonies (see below), only counts at day 10 are presented. Unlike bone marrow, colony formation by lung mononuclear cells required the presence of methylcellulose and higher cell densities. Cultures were routinely established with 4 x 105 cells in a 1-mL volume, in triplicate. Colonies derived from lung cells grew more slowly than those from bone marrow, but their morphology resembled eosinophil colonies derived from bone marrow.2
As a rule, counting at day 10 yielded higher counts of lung-derived colonies than at day 7, due to improved colony detection with well-preserved colony morphology. Results always refer to counts at day 10. For typing, individual colonies were aspirated with a micropipette under the inverted microscope, were transferred to microscope slides, and were stained (Panoptic stain or EPO). Results are calculated as the mean ± SEM of progenitor numbers per animal, based on the number of colonies formed by a standard inoculum (lung, 4 x 105 cells; BAL and spleen, 2 x 105 cells) and on the number of mononuclear cells recovered per animal in each cell source. For blood, the results are presented per milliliter of blood. For all sources of hemopoietic cells, liquid cultures (106 cells in a 1-mL volume, in a 24-well cluster2
) were seeded in RPMI medium/10% FCS, 2 mmol/L L-glutamine, and penicillin-streptomycin at 37°C, in an atmosphere of 5% CO2/95% air, at least in triplicate, in the presence of GM-CSF (2 ng/mL), IL-3 (2 ng/mL), and IL-5 (1 ng/mL). The frequency of EPO+ cells was determined at day 7.2
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Results and Discussion
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Significantly higher numbers of low-density mononuclear cells were recovered from sensitized/challenged mice than from naive controls, with an approximate 10-fold increase in mice that were challenged repeatedly with OVA. Very few or no colony-forming cells were present among lung mononuclear cells from naive mice. Sensitization by itself did not significantly increase progenitor numbers relative to naive controls. Progenitor numbers were significantly increased in mice sensitized following protocol 1 and challenged with OVA, relative to both naive controls (p = 0.003) and sensitized controls (p = 0.047). Repeated challenges of sensitized mice (protocol 2) did not significantly increased progenitor numbers relative to protocol 1. When lung mononuclear cells were separated into CD34+ and CD34- subpopulations, colonies were formed only from CD34+ cells. The frequency of colony-forming cells in the low-density mononuclear fraction from sensitized/challenged lung cells was roughly 1 in every 4,000 cells. This represents an approximate 100-fold enrichment over naive cells from BALB/c mouse lung. For comparison, myeloid progenitors responsive to GM-CSF represent about 1 in every 2,000 bone marrow cells for both naive and sensitized BALB/c mice in our routine culture conditions.4
Hence, hemopoietic progenitors were approximately one half as frequent in allergic lung as in allergic bone marrow. Eighty-four percent of all lung-derived colonies were EPO+, indicating a strong bias toward eosinopoiesis. No significant response to IL-5 was observed in liquid cultures from naive or from sensitized BALB/c mice in the absence of OVA challenge. In contrast, a vigorous response was seen in cultures from challenged, sensitized mice that was comparable in magnitude to that elicited in challenged, sensitized bone marrow.2
4
These findings, taken together, indicate that hemopoietic progenitors and precursors selectively ac-cumulate in the murine lung after allergen sensitization and challenge. BAL fluid from sensitized/challenged mice contained very small numbers of colony-forming cells, while large numbers of progenitors could be recovered from digested lung fragments of the same animals. All colonies from BAL were EPO-. This shows that progenitor accumulation in the lung cannot be estimated from the numbers of progenitors in BAL fluid. On the other hand, blood and spleen did contain colony-forming cells. The total number of colony-forming cells in the circulation (assuming a total blood volume of 2 mL for the mouse) amounted to two thirds of the total number of progenitors isolated from lung.
Hemopoietic cells isolated from allergic BALB/c mice lungs differed from those in the bone marrow of the same animals in terms of their sensitivity to pharmacologic modulators. As shown in Figure 1 , dexamethasone enhanced the responses of eosinophil precursors from bone marrow to IL-5 but inhibited the responses of eosinophil precursors from the lung under the same conditions. Because, in current models, the suppression of tissue eosinophilia is thought to underlie the therapeutic effects of steroids,4
hemopoietic cells accumulating in the lungs, unlike those in bone marrow, respond to dexamethasone as predicted from these models. This highlights potentially important differences between these hemopoietic populations.
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Figure 1.. The effect of dexamethasone (DEX) on the response of eosinophil precursors to IL-5. The data are given as the percentage of EPO+ cells in liquid culture (mean ± SEM) when hemopoietic cells isolated from the lung (top) or from the bone marrow (bottom) of OVA-sensitized and challenged BALB/c mice were cultured for 7 days in the presence of recombinant murine IL-5 (1 ng/mL), alone or in the presence of dexamethasone; concentration, 10-7 mol/L. * = significant differences (p < 0.01) relative to the respective IL-5 controls.
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Footnotes
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Abbreviation: EPO = eosinophil peroxidase; FCS = fetal calf serum; GM-CSF = granulocyte-macrophage colony-stimulating factor; IL = interleukin; IMDM = Iscove modified Dulbecco medium; OVA = ovalbumin
This research was supported by Institut National de la Santé et de la Recherche Médicale (INSERM)-Leprosy Care Outpatient Clinic of the Oswaldo Cruz Foundation (FIOCRUZ), Financiadore de Estudos e Pesquisas, Coordnaç
o de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional do Desenvolvimento Cientifico e Tecnológico, and the Institut Pasteur.
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References
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- Robinson, DS, Damia, R, Zeibecoglou, K, et al (1999) CD34(+)/interleukin-5R- messenger RNA+ cells in the bronchial mucosa in asthma: potential airway eosinophil progenitors. Am J Respir Cell Mol Biol 1999 20,9-13
- Gaspar, Elsas MI, Joseph, D, Xavier, Elsas P, et al Rapid increase in bone marrow eosinophil production and responses to eosinopoietic interleukins triggered by intranasal allergen challenge. Am J Respir Cell Mol Biol 1997;17,404-413
- Horton, MA, Larson, KA, Lee, JA, et al Cloning of the murine eosinophil peroxidase gene (mEPO): characterization of a conserved subgroup of mammalian hematopoietic peroxidases. J Leukoc Biol 1996;60,285-294
- Gaspar, Elsas MI, Maximiano, ES, Joseph, D, et al Upregulation by glucocorticoids of responses to eosinopoietic cytokines in bone-marrow from normal and allergic mice. Br J Pharmacol 2000;129,1543-1552
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Abstract
Introduction
