Deficiency of plasminogen receptor, Plg-RKT , causes defects in plasminogen binding and inflammatory macrophage recruitment in vivo.

Essentials Plg-RKT is a novel integral membrane plasminogen receptor. The functions of Plg-RKT in vivo are not known. Plg-RKT is a key player in macrophage recruitment in the inflammatory response in vivo. Plg-RKT deficiency is not compatible with survival of the species. SUMMARY: Background Plg-RKT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-RKT in vivo we generated Plg-RKT-/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-RKT deletion on reproduction, viability, health and spontaneous thrombosis and inflammation. Results Plg-RKT-/- mice were viable and fertile. Survival of Plg-RKT-/- mice and Plg-RKT+/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-RKT-/- females died within 2 days of birth, consistent with a lactation defect in Plg-RKT-/- mothers. Additionally, there was a significant effect of Plg-RKT deficiency on the growth rates of female, but not male, mice. In experimental peritonitis studies, Plg-RKT-/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-RKT-/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-RKT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-RKT deficiency is not compatible with survival of the species, due to the death of all offspring of Plg-RKT-/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo.

Formation of immunodeficiency in newborn mice exposed to nicotine during intrauterine development.

Exposure to nicotine during intrauterine development leads to immunodeficiency manifested in inhibition of delayed-type hypersensitivity reaction and reduced number of antibody-producing cells forming in response to sheep erythrocytes in newborn mice. The number of splenic CFU in the bone marrow of newborn mice exposed to nicotine in utero is decreased compared to the control. By contrast, nicotine induced an increase in splenic CFU count in fetal liver. We concluded that nicotine modifying the hemopoietic microenvironment delayed the release of primitive precursors from fetal liver, which impaired colonization of fetal bone marrow and led to imbalance in the production of mature blood cell, including immune system cells.

Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.

Tissue- and epitope-specific mechanisms account for the diverse effects of anti-CD44 antibodies on the maintenance of primitive hematopoietic progenitors in vitro.

The identification of rare stromal cells that support high levels of stem cells has opened avenues to identify molecules that contribute to the maintenance of these cells. We show that the maintenance of long-term culture initiating cells (LTC-IC) in stromal cell-supported cultures can be modulated via mAbs specific for CD44. mAb IM7.8.1 suppressed while mAb RAMBM44 enhanced LTC-IC levels in culture. Genetic polymorphisms in CD44 were used to show that the stromal cell compartment is targeted by mAb RAMBM44 and the hematopoietic compartment by mAb IM7.8. Neither of the CD44-specific mAbs inhibited adhesion of LTC-IC to the stroma, suggesting alternative mechanisms of action. In support of this interpretation, we show that mAb RAMBM44 directly induces signal transduction in the stromal cell line S17 but not in hematopoietic cells. Conversely, mAb IM7.8 elicited the appearance of phosphorylated bands in hematopoietic cells, but not in stromal cells. Collectively, the data indicate that the opposing effects of CD44-mediated regulation can be explained by different cellular programs that are elicited in distinct cell compartments. The binding of the enhancing mAb RAMBM44 to CD44 is specifically inhibited by collagen IV, while binding of the suppressive mAb IM7.8.1 is inhibited by a substance contained in the supernatant of the stromal cell line AC3.U. Thus, the CD44 epitopes defined by the mAbs bind distinct ligands and the ligands provide a potential physiological counterpart for the regulatory actions of the mAbs.

Hyaluronate-enhanced hematopoiesis: two different receptors trigger the release of interleukin-1beta and interleukin-6 from bone marrow macrophages.

The glycosaminoglycan hyaluronate (HA) is part of the extracellular environment in bone marrow. We show here that HA activates signal transduction cascades important for hemopoiesis. In myeloid and lymphoid long-term bone marrow cultures (LTBMC), treatment with hyaluronidase (HA'ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown. HA induces bone marrow macrophages to secrete IL-1beta (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal transduction pathways: CD44 links to a pathway activating p38 protein kinase while the other yet unknown receptor induces Erk activity. There was no difference of the effect of HA and HA'ase on hematopoiesis in LTBMC and on cytokine production by macrophages in CD44-deficient mice compared with wild-type mice, indicating that the CD44 hyaluronate receptor and its signal transduction can be compensated for. Our data suggest a regulatory role for the extracellular matrix component HA in hematopoiesis and show the induction of signal transduction by HA receptors.

Involvement of CD44 variant isoform v10 in progenitor cell adhesion and maturation.

CD44 has been described repeatedly to be involved in hematopoiesis. Here, we addressed the question of functional activity of CD44 variant isoform v10 (CD44v10) in progenitor cell maturation by in vivo and in vitro blocking studies with a monoclonal antibody and a receptor globulin. We became interested in this question by the observation that CD44v10 is expressed, although at a low level, on a subpopulation of bone marrow cells. Flow cytometry revealed that 15%-20% of hematopoietic cells in the fetal liver and 25%-35% of bone marrow cells in adult mice were CD44v10 positive. The majority of CD44v10+ cells was HSA+/J11d+ and CD43+. CD44v10 was not detected on CD4+, CD8+, IgM+, or IgD+ cells. A CD44v10 receptor globulin did not bind to hematopoietic progenitor cells, but to stromal elements. The CD44v10-CD44v10 ligand interaction had a major impact on the adhesion of progenitor cells to stromal elements. When healthy animals received repeated injections of either anti CD44v10 or the CD44v10 receptor globulin, committed progenitors were mobilized and significantly augmented numbers were recovered in the spleen and the peripheral blood. Furthermore, the CD44v10-CD44v10 ligand interaction, which had no impact on progenitor expansion, influenced progenitor maturation, particularly of the B-cell lineage. Although the nature of the CD44v10 ligand remains to be explored, the supportive role of CD44v10 in progenitor maturation and, importantly, the efficient mobilization of progenitor cells by anti-CD44v10 and a CD44v10 receptor globulin could be of clinical benefit in peripheral blood stem cell transplantation.

Two different functions for CD44 proteins in human myelopoiesis.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Functional activity of CD44 isoforms in haemopoiesis of the rat.

Expression of CD44 is involved in the maturation as well as the homing of haemopoietic progenitor cells. Whether these processes are mediated by CD44 standard (CD44s) or variant (CD44v) isoforms is unknown. To assign functional activities of CD44 in haemopoiesis of the rat to distinct isoforms, ligand binding of haemopoietic progenitor cells was inhibited by monoclonal antibodies recognizing an epitope on CD44s (Ox50) or CD44 exon v6, (1.1ASML). The vast majority of rat bone marrow cells (BMC) as well as stromal cells and non-adherent cells in long-term bone marrown culture (LTBMC) expressed CD44s. Bone marrow cells and non-adherent cells in LTBMC, but not the stromal cells, also contained a population of large and granulated cells, which stained with anti-CD44v6. In vivo and in vitro reconstitution experiments revealed that homing of BMC as well as settlement on stromal elements was influenced exclusively by anti-CD44s, which also inhibited proliferation of progenitor cells. Anti-CD44v6 had no influence on homing and seeding, but interfered with stroma formation and progenitor maturation. Finally, restoration of functional activity of T-lineage cells was impaired in the presence of anti-CD44v6. The data indicate that CD44s and CD44v6 fulfilled distinct functions in haemopoiesis of the rat. Although CD44s facilitated homing and expansion of stem cells, progenitor cells, CD44v6 was involved in differentiation processes, particularly of lymphoid progenitor cells.

Requirement for CD44 in proliferation and homing of hematopoietic precursor cells.

The hematopoietic form of the adhesion molecule CD44 is known to be involved in lymphocyte maturation and homing. To define lineage and stage of maturation, which requires expression of CD44, murine long-term bone marrow cultures were established. Stroma formation and proliferation of early as well as committed erythroid, myeloid, and lymphoid progenitors were evaluated under the influence of monoclonal anti-CD44 antibodies. Although the formation of stromal elements was not affected, formation of cobblestone areas was completely abolished. [3H]thymidine suicide confirmed that anti-CD44 treatment interfered with the proliferation of early and committed hematopoietic progenitor cells. In addition, homing and seeding of bone marrow cells was impaired by anti-CD44. The data are indicative of a dual functional mode of CD44 in adhesion and proliferation of hematopoietic progenitors and confirm an essential requirement for the molecule in early stages of hematopoiesis.

Expression of CD44 variant isoforms in peripheral blood leukocytes in malignant lymphoma and leukemia: inverse correlation between expression and tumor progression.

In a variety of human tumors, including high grade Non-Hodgkin's lymphoma (hgNHL), a linkage between expression of CD44 variant isoforms (CD44v) and tumor progression has been described. In search of an easily accessible diagnostic parameter, expression of CD44 standard (CD44s) and CD44 variant isoforms (exons v5, v6, v7 and v10) in peripheral blood lymphocytes (PBLs) of patients with hematological malignancies was evaluated by fluorescence activated cell scanning. The analysis of 30 blood samples of healthy donors and patients with non-malignant diseases and of 183 blood samples of patients with malignant hematological disorders revealed that only in patients with malignant disorders did a measurable proportion of PBLs express CD44 variant isoforms, mostly exons v5, v6, v7 and, less frequently, exon v10. Elevated levels of CD44v expression were noted in PBLs of patients with acute and chronic myeloid leukemia (AML: 16%, CML: 25%), Hodgkin's disease (HD: 17%), multiple myeloma (MM: 22%), polycythemia vera (PV: 33%), acute lymphoid leukemia (ALL: 23%) and, most frequently, in PBLs of patients with non-Hodgkin's lymphoma (NHL:54%). CD44v expression was not restricted to the malignant phenotype, but instead was also noted in T cells, B cells and monocytes, preferentially in a subpopulation of large cells. Furthermore, expression of CD44v in PBLs was not linked to the histological grading or clinical staging. There was, however, an inverse correlation with tumor progression, whereas response to therapy was frequently accompanied by upregulation of CD44v. Thus, expression of CD44v in the PBLs of patients with NHL mainly reflected immune responsiveness. Since NHL manifests itself primarily in lymphoid organs, its progression is difficult to follow. Monitoring of CD44v in PBLs could be used as an additional and convenient parameter for surveying the course of disease.

Endogenous retroviral envelope peptide expression is involved in a regulation of lymphocyte and hematopoietic precursor activity.

A biological function of endogenously expressed MuLV p15E-related proteins for lymphocyte and hematopoietic precursor activity in mice was examined. A high level of endogenous p15E-related peptide expression in spleen cells of mice with hemolytic anemia rendered by phenylhydrazine (PHZ) treatment was observed, detected by hyperimmune rabbit antisera against amino acid sequence which compose the immunosuppressive domain (ISD) of exogenous viral transmembrane (TM) p15E protein. The conditioned medium of these cultured cells (PHZ/CM) was inhibitory for lymphocyte blastogenesis and granulocyte-macrophage (GM) precursor activity, but stimulatory for the erythroid colony growth. When added to PHZ/CM, anti-ISD/p15E antibodies were capable to abrogate these effects. These antibodies bound 14K and 48K structural peptides contented in PHZ/CM as presumably smaller components of env gene products. Given together, the results indicate that erythroid immature cells produce proteins appearing in cell culture medium which exert p15E-related properties. These peptides are suggested to exert a down regulation for both lymphocyte and GM precursor activities, and the colony-promoting effect towards erythroid compartment cells.

Antisense oligonucleotide complementary to endogenous retroviral MCF env gene inhibits both BFU-E and CFU-S colony formation in mice.

A possible biologic activity of endogenously expressed env sequence of retroviral mink cell focus-forming virus (MCF) genome for hematopoietic colony formation was studied in mice. Antisense 20-mer complementary to MCF env sequence was used to detect the result of blockage of this gene translation on the potency of marrow cells to form colonies of erythroid (BFU-E), myeloid granulocyte-macrophage (CFU-GM), and stem cell (day 11 CFU-S) hematopoietic compartments. A large relative decrease in BFU-E number was found in bone marrow cell cultures preincubated with antisense oligonucleotide during 4 h, whereas CFU-GM colonies remained unaffected. A marked reduction of CFU-S colony formation was also registered under antisense oligomer influence. Following a decreased proliferation of erythroid progenitors, we suggest the mechanism by which antisense oligonucleotide could cause the loss of colony formation. Taken together, these data allow to propose that the expression of this gene is naturally significant for hematopoietic progenitor activity exerting some property of env gene products to regulate the growth of erythroid and multilineage hematopoietic precursors.

The influence of synthetic peptide from retroviral transmembrane protein p15E on murine spleen cell proliferation and bone marrow hemopoietic precursor colony formation.

The retroviral transmembrane p15E peptide is known to suppress a wide variety of immune cell functions, suggesting a role for immunosuppression associated with retroviral infection. The 10-amino acid sequence from the highly conserved portion of p15E (CKS-10) is capable of reproducing this inhibitory activity. In this study we set out to determine the influence of this decapeptide on murine spleen cell mitogen-induced proliferation and hematopoietic granulocyte-macrophage and erythroid precursor colony formation in vitro. A dose- and time-dependent suppression of spleen cell blastogenic response was produced by the CKS-10 peptide. When bone marrow cells were incubated with decapeptide, the significant decrease of CFU-GM colony number was also dose-dependent. In contrast, the same doses of CKS-10 peptide which induced a most significant inhibition of CFU-GM colony formation caused a marked increase of BFU-E colonies. A most pronounced effect of the peptide on bone marrow hematopoietic progenitor activity was produced by prolonged exposure to the peptide. Given the results of this study, it seems likely that, in addition to the cytopathic effect of retroviruses on the lymphocytes, viral peptide-mediated hematopoiesis disorders may also play an important role in the pathogenesis of immunodeficiency associated with retroviral infections.