Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches

In the bone marrow, the special microenvironment niches nurture a pool of hematopoietic stem cells (HSCs). Many HSCs reside near the vasculature, but the molecular regulatory mechanism of niches for HSC maintenance remains unclear. Here we showed that the induced deletion of CXCR4, a receptor for CXC chemokine ligand (CXCL) 12 in adult mice, resulted in severe reduction of HSC numbers and increased sensitivity to myelotoxic injury, although it did not impair expansion of the more mature progenitors. Most HSCs were found in contact with the cells expressing high amounts of CXCL12, which we have called CXCL12-abundant reticular (CAR) cells. CAR cells surrounded sinusoidal endothelial cells or were located near the endosteum. CXCL12-CXCR4 signaling plays an essential role in maintaining the quiescent HSC pool, and CAR cells appear to be a key component of HSC niches, including both vascular and endosteal niches in adult bone marrow.     

Pre-pro-B cell growth-stimulating factor (PPBSF) upregulates IL-7Ralpha chain expression and enables pro-B cells to respond to monomeric IL-7.

Although pro-B cells are well represented in IL-7 knockout (KO) mice, they express abnormally low concentrations of the interleukin-7 receptor alpha-chain (IL-7Ralpha) and do not generate pre-B cells. Here, we demonstrate that pro-B cells from IL-7 KO mice can be induced to generate pre-B cells and immature B cells by exposure to recombinant IL-7 (rIL-7) in vivo but not in vitro. Experiments in recombinant activation gene-1 (RAG-1) KO mice indicate that the in vitro unresponsiveness of IL-7(-/-) pro-B cells to rIL-7 is unrelated to the absence of a functional pre-B cell receptor (pre-BCR). Rather, it appears to be due to the suboptimal expression of the IL-7Ralpha chain. Thus, IL-7(-/-) pro-B cells readily respond to rIL-7 in vitro if IL-7Ralpha chain expression is first upregulated by exposure to IL-7 in vivo or to IL-7(+/+) bone marrow (BM) stromal cells or conditioned medium (CM) therefrom in vitro. Similar results were obtained when pro-B cells from IL-7 KO mice were cultured on IL-7(-/-) BM stromal cells in the presence of rIL-7. This suggested that the recently described pre-pro-B cell growth-stimulating factor (PPBSF), a self-assembling hybrid cytokine comprising IL-7 and the stromal cell-derived hepatocyte growth factor beta-chain (HGFbeta), is required to stimulate pro-B cells from IL-7 KO mice. This inference was verified by demonstrating that purified PPBSF upregulates IL-7Ralpha chain expression on IL-7(-/-) pro-B cells in vitro and enables them to respond to rIL-7 in a stepwise manner. We, therefore, postulate that PPBSF is the operative form of IL-7 that normally induces IL-7Ralpha(lo) pre-pro-B cells to proliferate and differentiate into IL-7Ralpha(hi) pro-B cells, which then proliferate and differentiate into pre-B cells on stimulation with monomeric IL-7.     

The bone marrow is not only a primary lymphoid organ: The critical role for T lymphocyte migration and housing of long‐term memory plasma cells

In immunology and anatomy textbooks the bone marrow is described as a typical “primary lymphoid organ” producing lymphoid cells independent of antigens. The hematopoietic bone marrow is largely age‐dependent organ with great anatomical and functional differences among various species. There are estimates that about 12% of all lymphoid cells in the human body are found in the bone marrow at any given time (2% in the peripheral blood). Enormous numbers of T lymphocytes migrate to the bone marrow and partly return later to the blood. Many of these lymphocytes are memory CD4⁺ and CD8⁺ T cells. A few days after immunization a wave of plasma cells and their precursors migrate to the bone marrow where they lose their migratory response to CXCL‐12 and CXCL9. There is a relative enrichment of CD19⁺ B cells in the bone marrow outnumbering those in the blood and secondary lymphoid organs. This is not due to local production. The proliferation and migration kinetics of these lymphoid cells in the bone marrow have to be studied in more detail as this is of major clinical relevance.     

The effects of microenvironment and internal programming on plasma cell survival

Two populations of plasma cells (PCs) are formed after immunization. A short-lived population in the spleen and lymph nodes provides rapid protection. A long-lived population, mainly in the bone marrow, provides lasting immunity. The mechanisms responsible for the differences in PCs life span remain largely unknown. The goal of the current study was to compare the intrinsic survival capacity of isolated short-lived (spleen) versus long-lived (bone marrow) PCs. We approached this question by using a previously established in vitro model that measures PC survival in a supportive stromal environment. Regardless of the tissue source or isolation time point after immunization, the two PC populations showed similar intrinsic ability to survive in vitro. To test differences in the stromal microenvironments, stromal cells from marrow, spleen or lymph nodes were evaluated for ability to support PCs survival. Survival of isolated PC was always greater when co-cultured with marrow stromal cells compared with those from spleen (or lymph node) despite the finding that IL-6, necessary for PC survival in culture, was secreted by all three stromal cell sources. Additionally, low expression of B-cell-activating factor belonging to the tumor necrosis factor-family was detected in all three stromal isolates. In contrast, marrow stromal cells were distinguished by cell-surface phenotype and CXC chemokine ligand (CXCL)12, IL-7 and stem cell factor expression. Although CXCL12 has been suggested as a possible survival factor for PC, addition or neutralization of CXCL12 had minimal effect on PC survival. We conclude the mechanisms regulating PC longevity appear extrinsically driven and marrow favored, but the factors that give marrow stromal cells a unique advantage remain unknown.     

Processing of CXCL12 by different osteoblast-secreted cathepsins

Hematopoietic stem and progenitor cells (HSPCs) are known to reside in specialized niches at the endosteum in the trabecular bone. Osteoblasts are the major cell type of the endosteal niche. It is well established that secreted proteases are involved in cytokine-induced mobilization processes that release stem cell from their niches. However, migratory processes such as the regular trafficking of HSPCs between their niches and the periphery are not fully understood. In the current study we analyzed whether osteoblast-secreted cysteine cathepsins are able to reduce the direct interaction of HSPCs with bone-forming osteoblasts. Isolated human osteoblasts were shown to secrete proteolytically active cysteine cathepsins, such as cathepsins B, K, L, and X. All of these cathepsins were able to digest, although with different efficacy, the chemokine CXCL12, which is known to be important for retaining HSPCs in their niches. Of the 4 identified cathepsins, only cathepsin X was able to reduce binding of HSPCs to osteoblasts. Interestingly, nonactivated pro-cathepsin X and mature cathepsin X did not interfere with HSPC-osteoblast interactions. Only pro-cathepsin X treated with dithiothreitol, which unfolds but does not lead to full maturation of cathepsin X, significantly reduced HSPC adhesion to osteoblasts. These observations argue for a role of the accessible cathepsin X prodomain in diminishing cell binding. Our findings strongly suggest that the cysteine cathepsins B, K, and L constitutively secreted by osteoblasts are part of the fine-tuned regulation of CXCL12 in the bone marrow, whereas pro-cathepsin X with its prodomain can affect HSPC trafficking in the niche.     

Role of metalloproteinases MMP-9 and MT1-MMP in CXCL12-promoted myeloma cell invasion across basement membranes

Malignant plasma cells in multiple myeloma home to the bone marrow (BM), accumulate in different niches and, in late disease, migrate from the BM into blood. These migratory events involve cell trafficking across extracellular matrix (ECM)-rich basement membranes and interstitial tissues. Metalloproteinases (MMP) degrade ECM and facilitate tumour cell invasion. The chemokine CXCL12 is expressed in the BM, and it was previously shown that it triggers myeloma cell migration and activation. In the present work we show that CXCL12 promotes myeloma cell invasion across Matrigel-reconstituted basement membranes and type I collagen gels. MMP-9 activity was required for invasion through Matrigel towards CXCL12, whereas TIMP-1, a MMP-9 inhibitor that we found to be expressed by myeloma and BM stromal cells, impaired the invasion. In addition, we show that the membrane-bound MT1-MMP metalloproteinase is expressed by myeloma cells and contributes to CXCL12-promoted myeloma cell invasion across Matrigel. Increase in MT1-MMP expression, as well as induction of its membrane polarization by CXCL12 in myeloma cells, might represent potential mechanisms contributing to this invasion. CXCL12-promoted invasion across type I collagen involved metalloproteinases different from MT1-MMP. These data indicate that CXCL12 could contribute to myeloma cell trafficking in the BM involving MMP-9 and MT1-MMP activities.     

Origins of peripheral B cells in IL-7 receptor-deficient mice

The interleukin 7 (IL-7) signaling pathway is critical for early lymphoid differentiation. We found dramatic perturbations in fetal liver B cell development and confirmed a complete absence of developing B cells in the adult bone marrow in mice lacking the IL-7 receptor alpha (IL-7Ralpha) gene. We show that peripheral B-2 and B-1 cell populations are deficient in IL-7Ralpha-/- mice. B-2 follicular cell and peritoneal B-1 cell percentages are reduced, while B-2 marginal zone cell percentages are increased. A comparison of bone marrow and splenic populations at different ages revealed that the splenic B cell populations seen in adult IL-7Ralpha-/- mice first appear during neonatal development. We have measured N-nucleotide addition at the joints of V(D)J rearrangements in splenic B cells and have used it as a somatic marker to define and separate bone marrow-derived B cells from fetal liver-derived B cells. B cells isolated from the bone marrow and spleen of adult and neonatal IL-7Ralpha-deficient mice harbor high levels of N-nucleotide additions similar to those found in equivalent wild-type B cell populations. We conclude that the majority of splenic B cells in IL-7Ralpha-deficient mice originate from the bone marrow and not the fetal liver.     

Contribution of stromal cells to the migration, function and retention of plasma cells in human spleen: potential roles of CXCL12, IL-6 and CD54

Plasma cells (PC) localize to discrete areas of secondary lymphoid tissue and bone marrow (BM). The positioning of PC in different sites is believed to be regulated by chemokines and adhesion molecules expressed by accessory cells in the lymphoid tissue microenvironment. However, the mechanisms responsible for the positioning of PC within the red pulp (RP) of human spleen have not been elucidated. Therefore, we examined the contribution of human splenic stromal cells to the migration and function of human PC. Splenic PC expressed the chemokine receptor CXCR4 and responded to its ligand CXCL12. In contrast, PC lacked CXCR5 and CCR7, and consequently exhibited minimal migration towards CXCL13 and CCL21. Splenic stromal cells proved to be a rich source of CXCL12, and could induce the migration of human B cells. Furthermore, they supported Ig production by splenic PC mainly by secreting IL-6. Lastly, a striking difference between splenic and BM PC was the constitutive expression of CD11a by only splenic PC. Notably, splenic stromal cells expressed high levels of CD54, the counter-structure of CD11a, and splenic PC were positioned adjacent to stromal cells in the RP. Thus, we propose that stromal cells attract PC to the RP and contribute to their retention and function through the combined expression of CXCL12, CD54 and IL-6.     

Increased recruitment of bone marrow-derived cells into the brain associated with altered brain cytokine profile in senescence-accelerated mice

Bone marrow-derived cells enter the brain in a non-inflammatory condition through the attachments of choroid plexus and differentiate into ramified myeloid cells. Neurodegenerative conditions may be associated with altered immune-brain interaction. The senescence-accelerated mouse prone 10 (SAMP10) undergoes earlier onset neurodegeneration than C57BL/6 (B6) strain. We hypothesized that the dynamics of immune cells migrating from the bone marrow to the brain is perturbed in SAMP10 mice. We created 4 groups of radiation chimeras by intra-bone marrow-bone marrow transplantation using 2-month-old (2 mo) and 10 mo SAMP10 and B6 mice as recipients with GFP transgenic B6 mice as donors, and analyzed histologically 4 months later. In the [B6 → 10 mo SAMP10] chimeras, more ramified marrow-derived cells populated a larger number of discrete brain regions than the other chimeras, especially in the diencephalon. Multiplex cytokine assays of the diencephalon prepared from non-treated 3 mo and 12 mo SAMP10 and B6 mice revealed that 12 mo SAMP10 mice exhibited higher tissue concentrations of CXCL1, CCL11, G-CSF, CXCL10 and IL-6 than the other groups. Immunohistologically, choroid plexus epithelium and ependyma produced CXCL1, while astrocytic processes in the attachments of choroid plexus expressed CCL11 and G-CSF. The median eminence produced CXCL10, hypothalamic neurons G-CSF and tanycytes CCL11 and G-CSF. These brain cytokine profile changes in 12 mo SAMP10 mice were likely to contribute to acceleration of the dynamics of marrow-derived cells to the diencephalon. Further studies on the functions of ramified marrow-derived myeloid cells would enhance our understanding of the brain-bone marrow interaction.     

CXCR4–CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice

Antibody‐secreting cells (ASCs), including short‐lived plasmablasts and long‐lived memory plasma cells (LLPCs), contribute to autoimmune pathology. ASCs, particularly LLPCs, refractory to conventional immunosuppressive drugs pose a major therapeutic challenge. Since stromal cells expressing C‐X‐C motif chemokine‐12 (CXCL12) organize survival niches for LLPCs in the bone marrow, we investigated the effects of CXCL12 and its ligand CXCR4 (C‐X‐C chemokine receptor 4) on ASCs in lupus mice (NZB/W). Fewer adoptively transferred splenic ASCs were retrieved from the bone marrow of recipient immunodeficient Rag1^?/? mice when the ASCs were pretreated with the CXCR4 blocker AMD3100. CXCR4 blockade also significantly reduced anti‐OVA ASCs in the bone marrow after secondary immunization with OVA. In this study, AMD3100 efficiently depleted ASCs, including LLPCs. After two weeks, it decreased the total number of ASCs in the spleen and bone marrow by more than 60%. Combination with the proteasome inhibitor bortezomib significantly enhanced the depletion effect of AMD3100. Continuous long‐term (five‐month) CXCR4 blockade with AMD3100 after effective short‐term LLPCs depletion kept the number of LLPCs in the bone marrow low, delayed proteinuria development and prolonged the survival of the mice. These findings identify the CXCR4‐CXCL12 axis as a potential therapeutic target likely due to its importance for ASC homing and survival.     

MiR221 promotes precursor B‐cell retention in the bone marrow by amplifying the PI3K‐signaling pathway in mice

Hematopoietic stem cells and lineage‐uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B‐lineage committed preBI‐cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221‐controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B‐cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221‐enhanced PI3K signaling leads to increased expression of the anti‐apoptotic protein Bcl2 and VLA4 integrin‐mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI‐cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221‐expressing hematopoietic cells.     

A High-Fat Diet Increases IL-1, IL-6, and TNF-α Production by Increasing NF-κB and Attenuating PPAR-γ Expression in Bone Marrow Mesenchymal Stem Cells

It is well established that a high-fat diet (HFD) can lead to overweight and ultimately to obesity, as well as promoting low-grade chronic inflammation associated with increased levels of such mediators as TNF-α, IL-1, and IL-6. Bone marrow mesenchymal stem cells (MSCs), which are involved in hematopoietic niches and microenvironments, can be affected by these cytokines, resulting in induction of NF-κB and inhibition of PPAR-γ. Because this phenomenon could ultimately lead to suppression of bone marrow adipogenesis, we set out to investigate the effect of an HFD on the expression of PPAR-γ and NF-κB, as well as the production of IL-1, IL-6, and TNF-α in MSCs. Two-month-old male Wistar rats were fed a HFD diet and evaluated by means of leukograms and myelograms along with blood total cholesterol, triglyceride, and C-reactive protein levels. MSCs were isolated, and PPAR-γ and NF-κB were quantified, as well as IL-1, IL-6, and TNF-α production. Animals that were fed a HFD showed higher levels of blood total cholesterol, triglycerides, and C-reactive protein with leukocytosis and bone marrow hyperplasia. MSCs from HFD animals showed increased production of IL-1, IL-6, and TNF-α and increased NF-κB and reduced PPAR-γ expression. Therefore, ingestion of an HFD induces alterations in MSCs that may influence modulation of hematopoiesis.     

The tumor microenvironment: CXCR4 is associated with distinct protein expression patterns in neuroblastoma cells

In previous studies, we demonstrated that human neuroblastoma cells are equipped with the machinery to direct their homing to bone marrow. These tumor cells express the CXCR4 receptor for the bone marrow stroma-derived chemokine CXCL12 (SDF-1) and secrete the CXCL12 ligand. The present study was undertaken to explore possible differences in gene-expression patterns between neuroblastoma variants that over-express CXCR4 (designated STH cells) and those which express very little of this receptor (STL cells). The results of the study clearly indicate that these variants show a differential gene-expression profile. They differ in expression of some integrins such as VLA2, VLA3 and VLA6, of neuroendocrine-markers such as CD56 and synaptophysin, in the expression of c-kit and in the secretion of certain cytokines and growth factors such as TNFalpha, SDF-1, VEGF, IL-8, GM-CSF and IP-10. We hypothesize that these differences are due to an autocrine SDF-1alpha-CXCR4 axis.     

IL‐7 and immobilized Kit‐ligand stimulate serum‐ and stromal cell‐free cultures of precursor B‐cell lines and clones

Long‐term proliferating, D_HJ_H‐rearranged mouse precursor B‐cell lines have previously been established in serum‐ and IL‐7‐containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre‐B cells to undefined factors, hampering accurate analyses of ligand‐dependent signaling, which controls pre‐B cell proliferation, survival, residence and migration. Here, we describe a novel serum‐free, stromal cell‐free culture system, which allows us to establish and maintain pre‐B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit‐ligand, also called stem cell factor, produced as a kit‐ligand‐Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre‐B cells in an optimal density in plate‐bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre‐B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.     

Deregulation of the CXCL12/CXCR4 axis in methotrexate chemotherapy-induced damage and recovery of the bone marrow microenvironment

Cancer chemotherapy disrupts the bone marrow (BM) microenvironment affecting steady-state proliferation, differentiation and maintenance of haematopoietic (HSC) and stromal stem and progenitor cells; yet the underlying mechanisms and recovery potential of chemotherapy-induced myelosuppression and bone loss remain unclear. While the CXCL12/CXCR4 chemotactic axis has been demonstrated to be critical in maintaining interactions between cells of the two lineages and progenitor cell homing to regions of need upon injury, whether it is involved in chemotherapy-induced BM damage and repair is not clear. Here, a rat model of chemotherapy treatment with the commonly used antimetabolite methotrexate (MTX) (five once-daily injections at 0.75 mg/kg/day) was used to investigate potential roles of CXCL12/CXCR4 axis in damage and recovery of the BM cell pool. Methotrexate treatment reduced marrow cellularity, which was accompanied by altered CXCL12 protein levels (increased in blood plasma but decreased in BM) and reduced CXCR4 mRNA expression in BM HSC cells. Accompanying the lower marrow CXCL12 protein levels (despite its increased mRNA expression in stromal cells) was increased gene and protein levels of metalloproteinase MMP-9 in bone and BM. Furthermore, recombinant MMP-9 was able to degrade CXCL12 in vitro. These findings suggest that MTX chemotherapy transiently alters BM cellularity and composition and that the reduced cellularity may be associated with increased MMP-9 expression and deregulated CXCL12/CXCR4 chemotactic signalling.     

Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.     

Platelet factor 4 increases bone marrow B cell development and differentiation

Platelet factor 4 (PF4) is a megakaryocyte-/platelet-derived chemokine with diverse functions as a regulator of vascular and immune biology. PF4 has a central role in vessel injury responses, innate immune cell responses, and T-helper cell differentiation. We have now discovered that PF4 has a direct role in B cell differentiation in the bone marrow. Mice lacking PF4 (PF4^?/? mice) had fewer developing B cells in the bone marrow beginning after the pre-pro-B cell stage of differentiation. In vitro, PF4 increased the differentiation of hematopoietic progenitors to B cell lineage cells, indicating that PF4 has a direct effect on B cell differentiation. STAT5 activation is essential in early B cell development and PF4 increased the phosphorylation of STAT5. Taken together, these data demonstrate that PF4 has an important role in increasing B cell differentiation in the bone marrow environment.     

Microvesicles derived from mesenchymal stem cells: Potent organelles for induction of tolerogenic signaling

The adult bone contains a number of distinct populations of stem cells, including haematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells and fibrocytes. While haematopoietic stem cells are required to provide a lifelong supply of blood cells it is thought that the other populations of stem cells play a role in tissue regeneration and potentially disease. The chemokine CXCL12 is produced constitutively in the bone marrow and, acting via CXCR4, is critical in maintaining HSPCs in a quiescent state and retaining all subsets of stem and progenitor cells in the bone marrow environment. The cytokine G-CSF, used clinically to mobilize haematopoietic stem cells for bone marrow transplants, activates the sympathetic nervous system and bone marrow macrophages to reduce the expression of CXCL12 by bone marrow stromal cells, thereby promoting the exit of haematopoietic stem cells from the bone marrow. Understanding the molecular mechanisms underlying G-CSF stimulated mobilization has led to development of CXCR4 antagonists as fast acting mobilizing agents for haematopoietic stem cells. Evidence now suggests that CXCR4 antagonists can similarly mobilize distinct subsets of progenitor cells, namely the endothelial progenitor cells and mesenchymal stem cells, but this requires conditioning of the bone marrow with VEGF rather than G-CSF.     

Conditioning response to granulocyte colony-stimulating factor via the dipeptidyl peptidase IV-adenosine deaminase complex

G-CSF is routinely used to mobilize hematopoietic stem cells (HSCs) from bone marrow (BM) into peripheral blood before aphaeresis, but HSC harvesting can be suboptimal. On the other hand, transplanted HSCs sometimes fail to engraft a recipient BM microenvironment when G-CSF is used after transplantation, as pushing-CSF will push HSCs away from marrow. So, G-CSF action needs to be potentiated by other drugs. Marrow stromal cells establish a local CXCL12 concentration gradient that is the primary homing signal for HSCs. Pharmacological interventions that modify this gradient, therefore, have potential to help HSC mobilization (by decreasing CXCL12) and engraftment (by increasing CXCL12). CXCL12 inactivation is primarily mediated by dipeptidyl peptidase-IV. We review here the currently available drugs affecting this enzyme that could be used in the clinic to achieve phase-specific help for G-CSF.     

In vivo expression of interleukin 5 induces an eosinophilia and expanded Ly-1B lineage populations

In vitro, murine interleukin 5 (IL-5) acts as a colony-stimulating factor for eosinophils and induces B cells to proliferate and secrete antibody. In order to assess the biological effects of IL-5 in vivo, we transplanted lethally irradiated mice with bone marrow cells infected with a recombinant retrovirus bearing the IL-5 coding sequence. Within 2 weeks the peripheral blood of recipient mice exhibited a marked eosinophilia which persisted for at least 12 months, and an excess number of eosinophils was also evident in the bone marrow, spleen, liver, lung, and gut. Although no changes could be detected in the conventional B lymphocyte population, the peritoneum was replete with B cells characteristic of the Ly-1 lineage. Despite these expanded cell populations, mice remained healthy for 12 months after transplantation. These results suggest that IL-5 acts primarily on eosinophils and B cells of the Ly-1 lineage and that persistent overproduction of these cell types is not pathogenic.