Basal and induced granulopoiesis in outbred,F 1 hybrid and inbred mice: can inbreeding depression influence the experimental practice?

Introduction

Deleterious effects of inbreeding on fitness in a wide spectrum of mammalian species, including humans, have been often reported (e.g. refs ^1–3 ). Among the parameters of fitness discussed in the literature, those of blood and/or hematopoiesis seldom appear. Inbreeding depression in populations with high inbreeding levels was reported to be responsible for disorders such as immunocompetence in lemurs, ⁴ abnormalities in blood leukocyte and lymphocyte counts in robins ⁵ or in neutrophil dysfunction in rabbits. ⁶ Only episodic information is available on the functional differences in hematopoiesis between various mouse strains differing in their breeding status; remarkable differences in serum levels of a number of cytokines including granulocyte colony-stimulating factor (G-CSF) between an outbred and an inbred mouse strain with experimentally induced sepsis were reported. ⁷ Some basic peripheral blood parameters in various outbred and inbred mice were summarized in ref. ⁸ However, we found no paper reporting the influence of inbreeding on cell proliferation and differentiation in the hematopoietic organs. In this study, we compared the values of selected basic parameters of granulopoiesis among various inbred, outbred and F₁ hybrid mouse strains. Furthermore, we tested the ability of the hematopoietic system in selected inbred, outbred and F₁ strains of mouse to react to both non-specific stress (injection of saline) and specific stimulus (administration of a granulopoiesis-stimulating agent, meloxicam). Meloxicam is an inhibitor of cyclooxygenase-2 synthesis, which acts by inhibiting the production of prostaglandins that inhibit granulopoiesis. ^9,10 The positive influence of meloxicam on granulopoiesis was demonstrated previously. ^11,12

Materials and methods

All mice used in the study, i.e. male outbred ICR and NMRI mice, inbred C57BL6, C57BL10, BALB/c and CBA mice, as well as hybrid (CBAxC57BL10)F₁ mice aged three months and weighing an average 30 g were obtained from the breeding facility of the Medical Faculty, Masaryk University, Brno, Czech Republic, and represent the whole spectrum of mouse strains produced by the facility. Mice from a single source were used to exclude the possibility of false interstrain differences stemming from different breeder conditions. The use and treatment of the animals followed the European Community Guidelines as accepted principles for the use of experimental animals. All experiments were performed with the approval of the Institute's Ethical Committee.

Meloxicam (Sigma, St Louis, MO, USA) was dissolved in sterile saline and administered intraperitoneally at a dose of 20 mg/kg in a volume of 0.2 mL of saline. Another group of mice were treated with 0.2 mL of saline. Untreated animals were used as controls.

Concentrations of G-CSF in mouse serum were determined using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) in control animals or six hours after administration of meloxicam. This time interval was chosen on the basis of a previous study. ¹² The numbers of nucleated cells of the femoral marrow were determined using a Coulter Counter (Model ZF, Coulter Electronics, Luton, UK). Granulocyte–macrophage colony-forming cells (GM-CFC) were assayed using a standard semi-solid plasma clot technique. ¹² For evaluating the compartment of morphologically recognizable granulocytic precursor cells, differential counts were performed on marrow smears stained by the May Grünwald–Giemsa method. In the granulocytic lineage, myeloblasts through myelocytes were designated as proliferative cells, and metamyelocytes through segmented stages as non-proliferative cells.

Results are given as means ± standard error of mean. One-way analysis of variance and Tukey's post hoc test were used for statistical evaluations.

Results

In addition, it should be noted that these data do not show any relationship between the basal control values of G-CSF and GM-CFC numbers or proliferating cell counts. Assuming the causality between G-CSF levels and the cellular responses, the decisive regulatory inputs inducing cellular response seem to be the relative increases in G-CSF concentration, regardless of the absolute values reached after the stimulatory treatment.

Discussion

Interstrain differences in hematological parameters and functions in mice were frequently investigated. These studies used different inbred strains of mice and were aimed at solving questions of the genetical conditionality of hematopoietic functions. ^13–18 As shown in the Introduction, studies dealing with the functional hematopoietic effects of inbreeding and outbreeding are rare. ⁷ From this point of view our data present novel findings and provide the first evidence for the effects of inbreeding on processes of cell proliferation in the compartments of granulopoietic progenitor and precursor cells in mice. They suggest that outbred strains of mice may have a better ability to generate granulocytic cells, representing an important step towards realization of a non-specific immune response. On the contrary, the data obtained from the inbred strains investigated imply a lower magnitude of response to stimulation in their granulopoietic systems. It can be hypothesized from these findings that the phenomenon of inbreeding depression, including a decreased fitness, is expressed in the granulopoietic system of these mice.

Many animal investigations, e.g. transplantation studies or studies on the immune response to specific antigens, must be performed on inbred strains. Nevertheless, our results suggest that, contrary to the present custom of using inbred strains of mice, many studies aiming to evaluate the outcomes of pharmacological interventions on hematopoietic tissues, which are often carried with inbred strains of mice, ¹⁹ should consider the use of outbred mice, as they might represent a better model for such studies. Besides the more pronounced response of outbred mice to pharmacological stimuli, these animals are closer to the natural conditions, including those in human populations, in comparison with inbred strains. Studies using both inbred and outbred strains in parallel might be helpful in making a decision as to whether this suggestion should be applied in experimental practice.

Author contributions: MH and MP designed the research and wrote the paper; LD carried out the statistical evaluations; JH, ZH and LW carried out the experiments.