Aggregated IgE mimic interleukin-3-induced histamine synthesis by murine hematopoietic progenitors

Similar to interleukin-3 (IL-3), IgE acts on murine bone marrow cells by inducing histamine production. This effect does not result from degranulation of histamine-containing cells, but from histamine synthesis, as assessed by the following findings. (1) The histamine content of freshly isolated bone marrow cells is too low to account for the increase in extracellular histamine levels. (2) Neither IL-3 nor IgE induced histamine production in the presence of the specific inhibitor of histidine decarboxylase (HDC), the histamine-forming enzyme. (3) Both the enzymatic activity and the mRNA expression of HDC were enhanced in response to IL-3 or IgE. Artificial aggregation or formation of IgE immune complexes augmented ther effect on histamine synthesis, indicating that the aggregated form is responsible for this biologic activity. Yet, it is apparently not mediated by Fc epsilon RI because their cross-linkage by dinitrophenyl bovine serum albumin after presensitization with IgE did not induce histamine production by hematopoietic progenitors. Among other aggregated isotypes tested, only IgG2a and, to a lesser extent, IgG1 had a consistent but lower effect, whereas IgM and IgA were completely inactive. The target cells of IL-3 and IgE in terms of histamine synthesis do not belong to mature bone marrow populations, especially mast cells. They copurify with hematopoietic progenitors in the low-density layers of a discontinuous Ficoll gradient where they represent around 5% of the cells, as determined by in situ hybridization. This percentage remained the same, regardless of whether the cells were stimulated by IgE or IL-3 alone or by a combination of both, suggesting a common responder cell. In accordance with this notion, histamine-producing cells could not be distinguished from each other on the basis of density, size and internal structure, or rhodamine (Rh) retention. Finally, the effect of IgE is not caused by the induction of IL-3 because anti-IL-3 antibodies did not abrogate the effect of IgE.     

Peyer patches are not required for acute graft-versus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is a multistep disease process following allogeneic bone marrow transplantation (BMT). It has been postulated that the induction of acute GVHD requires the presence of Peyer patches (PPs). A new tumor necrosis factor (TNF)-deficient strain has been developed that totally lacks PPs and displays the defects characteristic of TNF ablation but not lymphotoxin-associated defects characterized by lack of both PPs and lymph nodes. To determine the necessity of PPs in acute lethal GVHD induction, we transplanted full major histocompatibility complex (MHC)-mismatched grafts into myeloablated TNF knockout recipients. No differences in the survival or GVHD-associated histopathologic lesions were observed between the recipients. We conclude that neither PPs nor host TNF-alpha is required for the development of acute lethal GVHD in mice that undergo myeloablative conditioning and allogeneic BMT.     

Mast cells are not essential to inflammation in murine model of colitis

A simple rat model of chronic intestinal inflammation was adapted to mice in order to ascertain whether mast cells play an essential role in its induction or perpetuation. Colitis was induced in C57BL mice by intrarectal administration of trinitrobenzene sulfonic acid in 50% ethanol. Higher doses of trinitrobenzene sulfonic acid per gram of body weight were required in mice than rats, with a narrower effective dose range (the upper dose limited by unacceptable mortality and the lower by decreased inflammation). Colons of treated mice were macroscopically inflamed, with transmural damage, adhesions to adjacent structures, and ulcerations. Inflammation was scored subjectively and by tissue weight and myeloperoxidase content; each index was increased dose-dependently by trinitrobenzene sulfonic acid doses of 0.3–10 mg. Six milligrams of trinitrobenzene sulfonic acid induced reproducible inflammation for up to four weeks. Trinitrobenzene sulfonic acid could induce inflammation in both mast-cell-deficient W/W^v mice and their normal +/+ littermates in a similar fashion. Thus it is possible to induce chronic colitis in the mouse. Mast cells are not essential participants in this process.     

Long-term culture of B lymphocytes and their precursors from murine bone marrow.

Growth of mature B cells and their precursors from mouse bone marrow was maintained in culture for greater than 1 year. Feeder layers of adherent bone marrow cells, established in medium containing fetal calf serum and no exogenous steroids, were used to provide an in vitro environment that supported continuous growth and development of these cells. In such bone marrow cultures, the number of cells bearing membrane immunoglobulin increased gradually for 4 wk and then decreased. Between 10 and 14 wk, some of the cultures gave rise to continuously dividing B-cell populations that contained pre-B cells (producing mu heavy chains only and sensitive to transformation by Abelson murine leukemia virus) as well as mature B cells (synthesizing both light and heavy chains of IgM). Immunoglobulin molecules synthesized by cells in these populations were heterogeneous in two-dimensional gel analysis. This suggests that mature B cells arose via maturation of pre-B cells in the cultures that involved rearrangement and expression of different variable region gene segments.     

Identification of committed mononuclear precursors for osteoclast-like cells formed in long term human marrow cultures

Nonadherent marrow mononuclear cells enriched for hematopoietic progenitor cells were cultured in semisolid medium with recombinant human granulocyte-macrophage colony-stimulating factor for 9 days to form colony forming unit-granulocyte macrophage (CFU-GM) colonies. 1,25-Dihydroxyvitamin D was then gently layered over the cultures. After 2 weeks, approximately 30% of the colonies that formed were composed of cells with a unique polygonal morphology. One hundred percent of the polygonal cells in these colonies crossreacted with the monoclonal antibody 23c6, which preferentially recognizes osteoclasts. Homogenous populations of these polygonal cells formed multinucleated cells (MNC) in suspension culture, 100% of which cross-reacted with the 23c6 monoclonal antibody, and greater than 90% of the MNC contracted in response to calcitonin. Approximately 20% of these MNC formed resorption lacunae on calcified matrices. These results suggest that 1) early osteoclast precursors are derived from CFU-GM, the committed granulocyte-macrophage progenitor; 2) committed mononuclear osteoclast precursors have a distinct polygonal morphology and cross-react with monoclonal antibodies that recognize mature osteoclasts; and 3) these mononuclear precursors are capable of forming multinucleated cells which fulfill the functional criteria for osteoclasts.     

Interleukin-6 is not involved in the interleukin-1-induced production of colony-stimulating factors by human bone marrow stromal cells and fibroblasts

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a role in regulation of hematopoiesis. Because IL-6 is coinduced with colony- stimulating factors (CSFs) by various cell types in response to stimulation with IL-1, we investigated whether IL-6 is involved in the IL-1-induced production of CSF by human bone marrow (BM) cells in long- term culture or human fibroblasts. We showed that IL-6 does not induce CSF production by these cells. Neither addition of exogenous IL-6 nor neutralization of endogenous production of IL-6 by an anti-IL-6 monoclonal antibody (MoAb) diminished the IL-1-induced colony- stimulating activity (CSA), indicating that IL-6 did not act synergistically with IL-1. Finally, IL-6 did not influence the kinetics of IL-1-induced CSA production by human fibroblasts. We conclude that IL-6, either alone or in combination with IL-1, does not induce CSF production by human BM stromal cells or fibroblasts.     

Osteoclast precursors in murine bone marrow express CD27 and are impeded in osteoclast development by CD70 on activated immune cells

Osteoclasts (OCs) are bone-resorbing cells that are formed from hematopoietic precursors. OCs ordinarily maintain bone homeostasis, but they can also cause major pathology in autoimmune and inflammatory diseases. Under homeostatic conditions, receptor activator of nuclear factor kappa-B (RANK) ligand on osteoblasts drives OC differentiation by interaction with its receptor RANK on OC precursors. During chronic immune activation, RANK ligand on activated immune cells likewise drives pathogenic OC differentiation. We here report that the related TNF family member CD70 and its receptor CD27 can also mediate cross-talk between immune cells and OC precursors. We identified CD27 on a rare population (0.3%) of B220⁻c-Kit⁺CD115⁺CD11b^low cells in the mouse bone marrow (BM) that are highly enriched for osteoclastogenic potential. We dissected this population into CD27^high common precursors of OC, dendritic cells (DCs) and macrophages and CD27^low/neg downstream precursors that could differentiate into OC and macrophages, but not DC. In a recombinant mouse model of chronic immune activation, sustained CD27/CD70 interactions caused an accumulation of OC precursors and a reduction in OC activity. These events were due to a CD27/CD70-dependent inhibition of OC differentiation from the OC precursors by BM-infiltrating, CD70⁺-activated immune cells. DC numbers in BM and spleen were increased, suggesting a skewing of the OC precursors toward DC differentiation. The impediment in OC differentiation culminated in a high trabecular bone mass pathology. Mice additionally presented anemia, leukopenia, and splenomegaly. Thus, under conditions of constitutive CD70 expression reflecting chronic immune activation, the CD27/CD70 system inhibits OC differentiation and favors DC differentiation.     

Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells

OBJECTIVE: The aim of this study was to determine whether the combination of a sizable generation of colony-forming cells (CFC) with the maintenance of their progenitors (pre-CFC) ensured by incubation in hypoxia is associated with a certain degree of cell cycling, ultimately responsible for "self-renewal" of pre-CFC. The effects of interleukin-3 (IL-3) on the cycling and CFC-generation potential of pre-CFC also was investigated. MATERIALS AND METHODS: In severely hypoxic (0.9% O(2)) murine bone marrow cell cultures containing stem cell factor, interleukin-6, and granulocyte colony-stimulating factor, pre-CFC maintenance was characterized by the culture-repopulating ability assay, an in vitro analogue of the marrow-repopulating ability assay. The proliferative history of CD34(+) cells in primary cultures was determined by PKH2 staining and related to their CFC-generation potential. In some experiments, subsets of CD34(+) cells sorted on the basis of the number of cell divisions (0, 1, >1) were independently characterized. RESULTS: In hypoxia, the numbers of CFC and CD34(+) cells were markedly reduced, whereas pre-CFC were maintained better than in air. Addition of 5-fluorouracil to hypoxic cultures for 2 days suppressed their CFC-generation potential. The CFC-generation potential of divided CD34(+) cells was maintained or increased with respect to that of undivided cells in hypoxia but not in air. IL-3 decreased CFC-generation potential at both oxygen concentrations. IL-3 also increased the number of CD34(+) cells that divided more than once in hypoxia, decreasing their CFC-generation potential. CONCLUSIONS: Maintenance of CFC-generation potential in hypoxia occurs mainly in a subset of cycling progenitors, despite their proliferation (self-renewal). IL-3 decreased CFC-generation potential by increasing the rate of pre-CFC proliferation beyond the first cycle, which probably results in their differentiation and loss of CFC-generation potential.     

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.     

Calcium ionophore but not phorbol ester promotes eicosanoids release by proliferating interleukin-3-dependent bone marrow cells

Eicosanoid release during multilineage hematopoiesis was assessed using freshly isolated mouse bone marrow cells cultured in the presence of interleukin-3 (IL-3) (10% WEHI-3 culture-conditioned medium). Cells that could release prostaglandin E2 (PGE2) when stimulated with calcium ionophore A23187, but not with phorbol ester (PMA), appeared within 4 days. The cells harvested on day 10 released 42 ng of PGE2/10(6) cells/mL after A23187 stimulation. Leukotriene B4 (LTB4) (4 ng/mL) was also detected after A23187 stimulation, but there was no detectable LTC4 (less than 0.5 ng/mL). Nonadherent bone marrow cells were isolated from 28-day cultures and cloned. All clones were strongly IL-3-dependent. Although other growth factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and CSF-1 failed to promote survival or support proliferation of the cells, three clones (11-1-A6, 3-2-D5, and 11-1-A1) showed significant increases in 3H-thymidine incorporation, respectively, after PMA treatment for 24 hours. Surviving cells displayed dominantly myeloid type morphology and phenotypic characteristics. The data suggest that IL-3 is important in the formation of PGE2-producing cells. In contrast to many macrophages (MO), neither the IL-3-dependent cell lines nor the IL-3-cultured bone marrow cells released significant amounts of PGE2 when stimulated with PMA or IL-3, although PMA and IL-3 both induced translocation of protein kinase C (PKC) to the membrane fraction. The lack of production of PGE2 and other eicosanoids by the PMA- and IL-3-stimulated cell lines was confirmed by measuring the release of 3H-arachidonic acid. The data suggest that in IL-3-dependent bone marrow cell lines the activation of eicosanoid metabolism requires elevated cellular Ca2+; PKC activation alone does not appear to be a sufficient stimulus.     

Interactions between NKT cells and Tregs are required for tolerance to combined bone marrow and organ transplants

We used a model of combined bone marrow and heart transplantation, in which tolerance and stable chimerism is induced after conditioning with fractionated irradiation of the lymphoid tissues and anti-T-cell antibodies. Graft acceptance and chimerism required host CD4(+)CD25(+) Treg production of IL-10 that was in-turn enhanced by host invariant natural killer (NK) T-cell production of IL-4. Up-regulation of PD-1 on host Tregs, CD4(+)CD25(-) conventional T (Tcon) cells, and CD8(+) T cells was also enhanced by NKT cell production of IL-4. Up-regulated PD-1 expression on Tregs was linked to IL-10 secretion, on CD8(+) T cells was linked to Tim-3 expression, and on CD4(+) Tcon cells was associated with reduced IFNγ secretion. Changes in the expression of PD-1 were induced by the conditioning regimen, and declined after bone marrow transplantation. In conclusion, NKT cells in this model promoted changes in expression of negative costimulatory receptors and anti-inflammatory cytokines by Tregs and other T-cell subsets in an IL-4-dependent manner that resulted in tolerance to the bone marrow and organ grafts.     

Characterization of murine hematopoietic progenitor subsets involved in interleukin-3-induced interleukin-6 production

Various murine cell populations were tested for their ability to generate interleukin-6 (IL-6) in response to IL-3. Among these, bone marrow cells exhibit the most prominent IL-6 production. The responder cells in this organ have been further characterized by cell fractionation on a discontinuous Ficoll gradient, fluorescence-activated cell sorting, and in situ hybridization. These procedures have allowed us to ascribe the following features to the cells mainly responsible for IL-3-induced IL-6 production: (1) they possess a low density and a relatively high forward and perpendicular light scatter (FLS/PLS); (2) they are characterized by a high rhodamine (Rh) retention; and (3) their enrichment in various subpopulations is similar to that obtained for progenitors forming colonies in the methylcellulose assay colony-forming units (CFU-C). In contrast, IL-3 target cells in terms of IL-6 production are absent both in the mature and in the most immature bone marrow compartment. Indeed, the Rh-dull population that is enriched for cells with marrow repopulating activity does not respond to the growth factor and mature cells cannot be induced to express IL-6 as assessed by (1) FLS/PLS characteristics, (2) the monoclonal antibody ER-MP 20 recognizing monocytes and granulocytic cells, and (3) in situ hybridization. Taken together, our data support the conclusion that the bone marrow cells generating IL-6 in response to IL-3 belong to a progenitor population with enhanced mitochondrial activity, comprising probably several types of immature cells of the myeloid lineage including macrophage/granulocyte precursors.     

Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6.

Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long-term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.     

Effects of human interleukin-3 on granulocytic colony-forming cells in human bone marrow

Human multilineage colony-stimulating factor (multi-CSF)/interleukin-3 (IL-3) induces colony formation from CFU-GEMM, BFU-E, and CFU-Eo when applied to in vitro cultures of highly enriched hematopoietic progenitor cells. No granulocytic colonies are formed in response to IL- 3. However, with appropriate assays, we demonstrate that IL-3 increases the size of G-CSF-induced granulocytic colonies; these colonies contain greater proportions of immature cells as compared with colonies stimulated by G-CSF alone. Furthermore, IL-3 promotes the survival of CFU-G in vitro, whereas in cultures not supplemented with IL-3, CFU-G extinguish within seven days. We conclude that IL-3, although it does not stimulate granulocytic colony formation by itself, regulates the survival and proliferative rate of granulocytic progenitors.     

Dendritic cells are required for effective cross-presentation in the murine liver

The liver harbors a diversity of cell types that have been reported to stimulate T cells. Although most hepatic dendritic cells are immature, a small population of CD11c(high) conventional dendritic cells (cDCs) exists that expresses high levels of costimulatory molecules. We sought to determine the relative contribution of cDCs to cross-presentation by the liver. In vitro, liver nonparenchymal cells (NPCs) depleted of cDCs induced only minimal proliferation and activation of antigen-specific CD8(+) T cells when loaded with soluble protein antigen. Using a transgenic mouse with the CD11c promoter driving expression of the human diphtheria toxin receptor, we found that selective depletion of cDCs in vivo reduced the number and activation of antigen-specific CD8(+) T cells in the liver after intravenous administration of soluble protein antigen. Adoptive transfer of DCs, but not CD40 stimulation, restored the hepatic T-cell response. Conclusion: Our findings indicate that the ability of the liver to effectively cross-present soluble protein to antigen-specific CD8(+) T cells depends primarily on cDCs. Despite costimulation, other resident liver antigen-presenting cells cannot compensate for the absence of cDCs.     

Culture of putative Langerhans cell bone marrow precursors: characterization of their phenotype

We searched for the presence of human CD1-positive cells in bone marrow populations in order to characterize putative Langerhans cell precursors. Bone marrow progenitors were cultured in 0.8% methylcellulose supplemented with 10% granulocyte-macrophage (GM) colony-stimulating factor(s) GCT and HTB9. We compared the kinetics of these two factors and found that GCT was the more appropriate for our study. After 8 days of culture, colony-forming units of granulocyte-macrophages (CFU-GM) were tested for the presence of CD1-positive cells using the immunofluorescence technique. Positive cells were counted by cytofluorometric analysis: 9.4% CD1a (BL6), 13.4% CD1c (L161), 4.3% CD1b (NuT2), 4.6% CD2 (T11), and 25.5% CD33 (My9). Ultrastructural features and phenotype were then specified by the immunogold labeling technique using electron microscopy. A subpopulation of CD1-positive cells showed the ultrastructural morphology of bone marrow pro-monocyte/monocyte cells. By using well-characterized monoclonal antibodies, it was demonstrated that these cells expressed the following phenotype: CD14+, CD33+, CD4+, HLA-DR+, HLA-DP+, HLA-DQ-, OKT10-, CD2-. These data indicate that these bone marrow promonocyte/monocyte progenitors express a phenotype similar to that of epidermal Langerhans cells but the density of each antigen is much lower than that observed on mature skin dendritic cells.     

Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage

Cartilage from normal controls, patients with osteoarthritis, and patients with rheumatoid arthritis produced no interleukin-6 (IL-6) in culture. However, IL-1 induced massive production of IL-6 (up to 135 ng/ml) in cartilage from all 3 sources, in a dose-dependent manner (in some cases, a peak value was reached). The levels of induced IL-6 were similar to those found in rheumatoid arthritis synovial fluid. At IL-1 concentrations that induced almost complete inhibition of proteoglycan (PG) synthesis, IL-6 production could still be increased considerably. Exogenous IL-6 inhibited PG synthesis by up to 25%. IL-1-induced inhibition of PG synthesis was reversed by antibodies against recombinant human IL-6. These results suggest that IL-6 is required for the IL-1-induced inhibition of PG synthesis.     

Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures

Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11b(bright), CD8alpha(-)) and "lymphoid-related" (CD11b(dull), CD8alpha(+)), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)-dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c(+) population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro-derived DCs, when treated with interferon-alpha or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8alpha, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti-interleukin-6 (IL-6) antibody, but not anti-GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.