Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration.

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Extramedullar B lymphopoiesis in liver schistosomal granulomas: presence of the early stages and inhibition of the full B cell differentiation.

Inflammatory granulomatous reactions in liver elicited by schistosomal infection have been shown to function as active extramedullar myelopoietic sites, producing potentially all the myeloid lineages. We have now addressed the question of the extramedullar B lymphopoiesis in these sites. We have shown the presence of early B cell precursors (pro-B cells) in the granulomas by immunophenotyping. Their total number in the liver was equivalent to the pro-B cells in the bone marrow of one femur. In agreement with their phenotype, the RT-PCR analysis showed that these cells expressed RAG-1 and lambda5 genes. However, the conversion of the pro-B to pre-B cells was not observed and no clonogenic B cell precursors could be detected in semi-solid cultures stimulated by IL-7. The granulomatous stroma was shown to produce IL-7 and express c-kit, and was able to sustain the full B lymphopoiesis in vitro. Conversely, the granuloma supernatant was shown to inhibit actively the development of B lymphocytes. We conclude that the granuloma environment elicits homing and proliferation of totipotent hematopoietic precursors, and that it is permissive for early commitment to the B cell lineage, but the full extramedullar production of B cell is abrogated by soluble factors produced inside the granulomas.

Murine schistosomiasis mansoni: experimental analysis of bone marrow and peripheral myelopoiesis.

In schistosomiasis a systemic hyperplasia of the monomacrophagic cell lineage is associated with its mild modifications in myelograms and hemograms. We monitored the in vitro proliferation of myeloid precursors obtained from bone marrow, blood, spleen, and liver. The macrophage colony-forming unit (M-CFU) numbers were stable in bone marrow but increased progressively in spleen and in liver, reaching in each organ the values equivalent to one femur. The bone marrow had an increased production and enhanced capacity to release M-CFU. Their quantitative increase in blood and in peripheral tissues of schistosome-infected mice was associated with their qualitative modifications: augmented proliferative capacity, enhanced adhesion, and accelerated differentiation. The accelerated release of monomacrophage progenitors and their enhanced proliferation in peripheral tissues potentially account for the relatively low involvement of the bone marrow and for an efficient in situ production of phagocytes, which participate in host reactions to parasites.

Haematopoietic capacity of colony-forming cells mobilized in hepatic inflammatory reactions as compared to that of normal bone marrow cells.

Chronic inflammatory periovular granulomatous reactions elicited in liver by schistosomal infection are a site of active myelopoiesis. We quantified the colony-forming cells (CFCs) in granulomas and found that the whole liver contains a number of CFCs roughly equivalent to 50% of a femur. Clonogenic analysis showed the presence of committed as well as pluripotent and totipotent CFCs. Long-term Dexter-type cultures showed that the granuloma-derived totipotent CFCs do not have self-renewal capacity. Hence, they did not correspond functionally to haematopoietic stem cells, despite the fact that the stroma established by adherent cells harvested from granulomas had the capacity to sustain long-term proliferation of bone-marrow-derived haematopoietic stem cells. We conclude that myelopoietic cytokines produced by inflammatory reactions in schistosomiasis elicit mobilization of bone marrow CFCs into the circulation, which can settle in hepatic granulomas. This environment may induce their proliferation and differentiation, but not their self-renewal, sustaining temporary production of myeloid cell lineages which nevertheless depends upon cell renewal from the bone marrow pool of haematopoietic precursors.

Increase of B-lymphocyte number and activity during experimental murine schistosomiasis mansoni.

1. In schistosomal infection, the hyperergic acute phase of the disease evolves progressively into the chronic one, with establishment of a relative equilibrium between the parasites and the corresponding host responses. This down-regulation of host reactivity is considered to be under the control of T-lymphocyte circuits. 2. In the present study, we investigated lymphocyte populations in spleens of normal mice and the kinetics of the B-cell number increase in mice in the acute, chronic and late chronic phases of schistosomal infection, and we monitored their proliferation and activity in antibody isotype secretion. 3. We observed polyclonal B-cell activation and modulation of Ig isotype production, compatible with the alternate predominance of TH2 and TH1 lymphocyte subsets, in the acute and the chronic phases of the disease, respectively.

Increase of B-lymphocyte number and activity during experimental murine schistosomiasis mansoni

1. In schistosomal infection, the hyperergic acute phase of the disease evolves progressively into the chronic one, with establishment of a relative equilibrium between the parasites and the corresponding host responses. This down-regulation of host reactivity is considered to be under the control of T-lymphocyte circuits. 2. In the present study, we investigated lymphocyte populations in spleens of normal mice and the kinetics of the B-cell number increase in mice in the acute, chronic and late chronic phases of schistosomal infection, and we monitored their proliferation and activity in antibody isotype secretion. 3. We observed polyclonal B-cell activation and modulation of Ig isotype production, compatible with the alternate predominance of TH2 and TH1 lymphocyte subsets, in the acute and the chronic phases of the disease, respectively.

Leishmania donovani surface glycoconjugate GP36 is the major immunogen component of the fucose-mannose ligand (FML).

Leishmania donovani promastigote glycoconjugate ligands, studied in our laboratory, that interact with the internalization receptors on BALB/c macrophages: the 'fucose mannose ligand' (FML), the 'phosphate mannogalactan ligand' (PMGL), and the 'lipopeptidephosphoglycan' (LPPD), interfered also with interaction between amastigotes and host cells in vitro. Among the three compounds studied, the FML was shown to be the most potent inhibitor of both promastigote and amastigote internalization, and to be present on parasite surface during the vertebrate-host cycle. The FML, but not the other two glycoconjugates, is a potent immunogen in rabbits (ELISA, agglutination and immuno-blots). Rabbit hyperimmune sera recognized essentially the 36 kDa band of FML. Mouse monoclonal antibodies against FML recognized either the 36 kDa or the 55 kDa band. No cross-reactivity between these two FML components was detected. No antigenic similarity could be detected between the 36 and 55 kDa bands of FML and the 'GP63' (promastigote surface proteinase) major surface leishmanial antigen. The 36 kDa-glycoprotein was identified as the major FML antigenic fraction and designated 'GP36'. The integrity of the glycidic moiety was necessary for its antigenicity. This L. donovani surface glycoprotein is apparently one of the major molecules involved in interactions between the parasite and the vertebrate host.

A glass wool-based method for purifying Trypanosoma cruzi trypomastigotes and identification of an epimastigote-specific glass-adherent surface peptide.

Glass wool, hydrophilic cotton wool, non-electrically charged BIO-GEL P2 and common tissue paper columns were used to purify trypomastigotes from a mixed Trypanosoma cruzi population grown in axenic culture medium. With all these columns, highly purified (up to 98%) trypomastigote preparations were obtained. Trypomastigote yields from cotton wool, BIO-GEL P2 and common tissue paper columns were not as high as from glass wool columns, from which yields varied from 69 to 80%. Purification on glass wool did not affect trypomastigote infectivity or virulence. Dead trypomastigotes could not be purified on glass wool columns. A glass-adherent amphiphilic peptide of 45 kDa, present in the cell membrane, was isolated from epimastigote but not from trypomastigote preparations.

Eosinophil granulocyte proliferation and differentiation in schistosomal granulomas are controlled by two cytokines.

In parasitic diseases, eosinophilia is controlled at the systemic level by soluble, circulating factors. In addition to their medullar production and migration to tissues involved by parasitosis, eosinophil populations in inflammatory infiltrates may be locally amplified by their in situ proliferation. In granulomas induced in liver tissue by eggs of schistosome worms, eosinophil proliferation and differentiation are observed. We have shown that they were under control of two cytokines, the activity of which can be demonstrated in supernatants of isolated granulomas maintained in culture for 24 hours. One of them has been identified as interleukin-5. The other one is secreted by adherent cells obtained from periovular granulomas, among which macrophages represent more than 99% of cells. It is considered to correspond to the previously described factor, secreted by inflammatory macrophages mobilized on intraperitoneal glass implants in mice with chronic schistosomiasis. In acute schistosomiasis, the activity of the interleukin-5 was predominant, whereas in the chronic phase of the disease, the stimulation of peripheral eosinopoiesis is taken over by the factor secreted by adherent cells. During the progression from the acute to the chronic phase of schistosomiasis, the immune reactivity of the host is down-regulated by T suppressor lymphocyte circuits. In addition, a redistribution of cellular controls of the host reaction to parasites may act as a complementary mechanism for establishment of the viable equilibrium between host and parasite.

In vitro induction of the fat-storing phenotype in a liver connective tissue cell line-GRX.

Liver connective tissue cells have been characterized as perisinusoidal myofibroblasts and hepatic lipocytes (Ito cells, fat-storing cells). A concept of a single mesenchymal cell population that may be modulated between these two phenotypes has been postulated. We have previously established a continuous murine cell line, GRX, obtained from fibrotic granulomatous lesions induced by schistosomal infection in mouse liver. This cell line is considered to represent liver myofibroblasts. In the present study we have induced the conversion of these cells into lipocyte (fat storing) phenotype by treatment with insulin and indomethacin. We have quantified the lipid synthesis and the increase of activity of involved enzymes during the induction of the fat-storing phenotype and described modifications of cell organization along this modulation of cell functions.

Experimental murine schistosomiasis mansoni: hyperplasia of the mono-macrophage cell lineage and stimulation of myeloid proliferation by peripheral macrophages.

1. Normal and schistosome-infected mice were similar in terms of the total number of bone marrow myeloid cell precursors and their proliferative capacity in vitro when stimulated with supernatants of L-929 cells containing M-CSF. 2. Delayed differentiation of bone marrow neutrophil granulocytes and blood monocytosis of infected animals were consistent with a modification in the differentiation of bone marrow myeloid precursors, favoring the production of a mono-macrophage cell lineage. 3. Macrophages isolated from periovular granulomas secreted a considerable stimulatory activity for the proliferation of the mono-macrophagic cell lineage, whereas peritoneal macrophages from the same animals had only a very low stimulatory activity. 4. We conclude that systemic hyperplasia of mono-macrophagic cells in schistosomiasis may be related to their increased release from the bone marrow and to their peripheral amplification in inflammatory tissue infiltrate as a consequence of the local production of stimulatory activity for their proliferation.

Experimental murine schistosomiasis mansoni: hyperplasia of the mono-macrophage cell lineage and stimulation of myeloid proliferation by peripheral macrophages

1. Normal and schistosome-infected mice were similar in terms of the total number of bone marrow myeloide cell precursors and their proliferative capacity in vitro when stimulated with supernatants ofL-929 cells containing M-CSF. 2. Delayed differentiation of bone marrow m=neutrophil granulocytes and blood monocytosis of infected animals were consistent with a modification in the differentiation of bone marrow myeloid precursors, favoring the production of a mono-macrophage cell lineage. 3. Macrophages isolated from periovular franulomas secreted a considerable stimulatory activity for the proliferation of the mono-macrophagic cell lineage, whereas peritoneal macrophages from the same animals had only a very low stimulatory activity. 4. We conclude that systemic hyperplasia of mono-macrophagic cells in schistomiasis may be relatted to their increased release from the bone marrow and to their peripheral amplification in inflammatory tissue infiltrate as consequence of the local production of stimulatory activity for their proliferation