Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice

Abstract

We have developed a transgenic mouse line, NJ.1638, which expresses high levels of IL-5 from T cells, with profound hematological consequences. Eosinophils comprise more than 60% of circulating white blood cells in these animals, with the total peripheral white blood cell counts increasing more than 40-fold relative to wild-type littermates. This extraordinary proliferative capacity is sustained by expanded sites of extramedullary hematopoiesis and is accompanied by multifocal, ectopic bone formation in the spleen. Histology of the splenic nodules revealed the presence of osteoid matrices and osteocytes trapped within mineralized trabecular plates. In addition, polarized light microscopy of calcified tissue sections revealed both woven bone and areas of organized lamellar bone. Morphometric assessments demonstrated that both the growth and mineralization of splenic bone occurred at rates nearly an order of magnitude higher than in skeletal bone. Skeletal bone metabolic parameters were also perturbed. We also observed heterotopic ossification of the spleen and perturbation of skeletal bone homeostasis following adoptive engraftment of transgenic marrow to wild-type recipients. These data suggest that IL-5 overexpression mediates bone formation through the mobilization of marrow-derived osteogenic progenitors and/or the inhibition of recruited osteoclasts.

Figure 1

Constitutive IL-5 expression induces pathological changes in the spleen. (a) Age-dependent cellularity changes in the spleens of wild-type and NJ.1638 mice. Single-cell suspensions were prepared from spleens of wild-type and transgenic mice at 1 month of age (wild-type [n = 3]; NJ.1638 [n = 6]) and 10 months of age (wild-type [n = 3]; NJ.1638 [n = 4]). Total cell counts (≥200) are represented as histograms and are expressed as group mean data (± SD). (b) Gross macroscopic comparison of the spleens from age- and sex-matched wild-type and NJ.1638 mice. The wild-type spleen is in the foreground. This comparison demonstrates the extreme splenomegaly and numerous white nodules/foci (arrows) that develop in adult NJ.1638 mice. The wet weight of the transgenic spleen (3.75 g) is more than 50 times that of the wild-type spleen. (c) Representative hematoxylin/eosin-stained section from a NJ.1638 spleen. This section exhibits a loss of normal spleen histology (i.e., white pulp and red pulp) and reveals the underlying ultrastructure of the white foci (f) appearing in b. Scale bar = 160 μm. (d) Higher magnification of the splenic nodule shown in c demonstrates the unique bone morphology of this structure including the presence of osteocytes (filled arrows) that occupy lacunae of the bone matrix. Scale bar = 20 μm.

Figure 2

The multifocal nodules of NJ.1638 spleens exhibit extensive staining for bone but not cartilage. (a) Dark-field photomicrograph of the wild-type and NJ.1638 spleens shown in Figure 1b after whole-mount staining with alizarin red/alcian blue to identify mineralized bone/cartilage, respectively. The wild-type spleen (arrow) displayed no staining with either reagent. In contrast, the numerous, and randomly distributed, nodules of the transgenic spleen stained heavily with alizarin red, providing evidence that these structures are centers of mineralization. Scale bar = 5 mm. (b) Higher magnification shows individual nodules exhibiting fine serpiginous extensions and a distinct lattice-like ultrastructure. Scale bar = 1 mm. (c) NJ.1638 splenic nodules represent areas of active bone formation/mineralization. Photomicrograph of a section from a transgenic spleen following Goldner’s staining. These nodules display patterns consistent with bone as characterized by the presence of newly formed unmineralized bone (i.e., red/orange staining osteoid [O]) and green-staining areas of mineralized bone matrix (Md). Scale bar = 50 μm. (d) High-magnification view of Goldner’s-stained nodules demonstrates the presence of both bone-forming osteoblasts (open arrows) at the periphery of the nodules, identified as relatively large cells with abundant basophilic cytoplasm and a pale stained nucleus. Osteocytes are seen as embedded cells within the mineralized matrix (filled arrows). Scale bar = 20 μm.

Figure 3

The SNs of NJ.1638 mice uniquely express an osteoblast marker and are areas of rapid/dynamic bone growth and mineralization. (a) Osteocalcin transcripts were detected in RNA recovered from NJ.1638 SNs. Total RNA from wild-type or transgenic spleen parenchyma and isolated mineralized SNs were reverse transcribed to generate template cDNA for PCR reactions using osteocalcin-specific primers (SSII+). RNA samples that were not reverse transcribed (SSII–) served as negative controls for genomic DNA contamination. Total RNA from NJ.1638 tibias and femurs served as a positive control sample for the osteocalcin primer set (294-bp RT-PCR amplicon indicated with an arrow). An actin primer set was used to demonstrate the integrity of all the samples being assayed (240-bp RT-PCR amplicon). DNA size standards (1-kb ladder) are indicated on the left. (b) NJ.1638 SNs are characterized by the presence of woven and lamellar bone. Evidence of matrix-embedded osteocytes and presence of woven bone were determined by examining toluidine blue–stained paraffin sections of SNs with polarized light. The birefringent areas of woven and lamellar bone result from polarized light interaction with collagen fibrils within the ossified nodule. Examples of regions of woven bone (Wo) and lamellar bone (Lm) are indicated. Scale bar = 0.1 mm. IL-5 expression leads to a generalized perturbation of bone metabolism including accelerated bone growth and mineralization in the spleen. (c) A representative fluorescence photomicrograph of a SN from a transgenic animal administered calcein (green fluorescence, arrow) and tetracycline (yellow fluorescence, arrowhead) at the beginning and end of a 10-day interval, respectively (see Methods). Scale bar = 0.1 mm. Label incorporation was assessed in bone samples from age-matched (5–8 months of age) wild-type (n = 6) and transgenic (n = 6) mice. These analyses are displayed graphically as histograms comparing the MAR (d) and the BFR/BS (e) of wild-type tibia and NJ.1638 SNs. Data are expressed as mean ± SEM. ^AP < 0.05.

Figure 4

Perturbations of bone growth occur in the long bones of NJ.1638 mice. (a) Comparison of cancellous/trabecular bone volume versus total bone volume (Cn.BV/TV%) of wild-type (n = 9) and NJ.1638 (n = 10) tibia. Data are expressed as mean ± SEM. ^AP < 0.05. Representative photomicrographs of hematoxylin/eosin-stained longitudinal sections of femurs from age- and sex-matched (8.5-month-old female mice) wild-type (b) and NJ.1638 (c) mice demonstrate that constitutive IL-5 expression leads to an increase in cancellous bone and occlusion of the marrow-containing lumen. Scale bars = 0.5 mm.

Figure 5

Splenic ossification and perturbations of skeletal long bone homeostasis occur in lethally irradiated wild-type mice after adoptive engraftment of NJ.1638 marrow. Representative photomicrographs of hematoxylin/eosin-stained decalcified sections from 8.5-month-old NJ.1638 spleen (a) and femur (c) are shown in comparison to sections of spleen (b) and femur (d) from irradiated wild-type mice 8–10 months after adoptive engraftment of NJ.1638 marrow. The striking histological similarities between the splenic and femoral ossification, regardless of the source animal, reflect both the likelihood of a single mechanism mediating bone formation as well as the marrow-derived nature of these osteogenic progenitors. Scale bars = 50 μm. Ct, cortical bone; Ma, bone marrow; EB, ectopic bone.