Peculiarities of immunocorrective effects of the bone marrow regulatory peptides (myelopeptides)

Myelopeptides (MPs) are low-molecular-weight immunoregulatory peptides of bone marrow origin. The peculiarities of their immunoregulatory effects are demonstrated with two of the six synthesized MPs, MP-1 (Phe-Leu-Gly-Phe-Pro-Thr) and MP-2 (Leu-Val-Val-Tyr-Pro-Trp). It is shown that MP action is directed to the damaged links of immunity. MP-1 enhances a decreased level of antibody production in cyclophosphamide (Cy)-treated mice, but does not influence the antibody formation in normal animals. MP-2 inhibits the tumor growth more in a tumor-bearing organism as the tumor size gets larger, insofar as MP-2 antitumor effect is concerned, by its ability to recover functional activity of T lymphocytes suppressed by tumor products. Selective immunocorrective effects of MPs are based on ligand-receptor interactions. Using FITC-labeled MP-1 and [3H]-labeled MP-2, specific binding of these peptides with appropriate cell populations is shown. The cytofluorimetric analysis revealed a target cell for MP-1--CD4+ T lymphocyte (T helper). The data obtained suggest that MPs are endogenic immunoregulators which participate in the maintenance of immune homeostasis.     

Endogenous immunoregulatory peptides (myelopeptides): structure, function, and mechanism of action

Previously unknown bioregulators from bone marrow, myelopeptides, were isolated, identified, and synthesized, and their biological properties and mechanism of action were studied in detail. Phe-Leu-Gly-Phe-Pro-Thr (MP-1) manifests an immunocorrecting effect by restoring the level of antibody production in animals suffering from immunodeficiencies of various etiologies; Leu-Val-Val-Tyr-Pro-Trp (MP-2) manifests an antitumor effect by abolishing the inhibitory action of tumor cells on the functional activity of T-lymphocytes; Leu-Val-Cys-Tyr-Pro-Gln (MP-3) stimulates phagocytosis by macrophages and, in this way, protects animals from infections caused by pathogenic microorganisms; and Phe-Pro-Arg-Ile-Met-Thr-Pro (MP-4) is a new factor of cell differentiation inducing terminal cell differentiation in the HL-60 and K-562 leukemic cell lines.     

Myelopeptide MP-5 and fluorescent derivatives: Synthesis and biological activity

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60–84% inhibition of growth of the following mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lymphocytes, but Ftc-MP-5 was practically inactive.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

Immunomodulating action of myelopeptides in severe closed experimental craniocerebral injury in rats

An immunocorrective effect of myelopeptides (MP) isolated from pig bone marrow cell culture supernatant in the early posttraumatic period in rats with severe cranial injury has been assessed. MP administration prevented cellular devastation of thymus and bone marrow, as well as spleen hyperplasia. The most marked MP effect was observed within the first 24 h after its administration. MP affected the functional and migration properties of both the entire population of lymphoid cells and individual subpopulations. MP had a pronounced protective effect against Staph. aureus infection during cranial trauma. MP completely prevented the death of animals and reduced more than 3-fold Staph. aureus persistence in the organism of animals. Anti-stress and protective effects of MP open vast prospects for their therapeutic and preventive application in the clinical practice.     

Effect of myelopeptide-1 on the functional activity of phagocytes from mice treated with cyclophosphane

The capacity of the bone marrow-derived myelopeptide-1 (MP-1) to affect in vivo and in vitro the functional activity of phagocytes of intact mice and mice treated with a cytostatic agent (cyclophosphane) has been studied. It was found that MP-1 produces a correcting effect on the functional activity of bone marrow and peripheral blood phagocytes. An optimal scheme of the injection of MP-1 to mice with the cyclophosphane-induced immunodeficiency was developed, which provides a maximum immunocorrecting action. MP-1 had the most pronounced effect on the quantitative characteristics and the functional activity of phagocytes of different localization when introduced prior to the cytostatic; under these conditions, the pep tide affects peripheral blood neutrophils. The results obtained enable one to consider MP-1 as a preparation protecting the peripheral blood phagocytes from the damaging action of cyclophosphane.     

Synthesis and properties of myelopeptides possessing differentiating activity

Bone marrow peptides Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro (MP-4) and Val-Asp-Pro-Pro (MP-6) have been synthesized by the classical and solid-phase methods of peptide chemistry, and their differentiating activity has been studied on leukemia cell lines HL-60 and K-562. It has been shown that both peptides induce the terminal differentiation of leukemic blasts; however, their mechanisms of action are different.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Regulation of fibronectin receptor distribution [published erratum appears in J Cell Biol 1992 Jul;118(2):491]

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b⁻ cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b⁻ cells increased the number of AlkP⁺ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b⁻ fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.     

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.     

Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells

Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.     

Colony stimulating activity of serum from germfree normal and leukemic mice

Serum from germfree Swiss/HaM mice exhibited a reduced capacity to stimulate granulocytic and mononuclear cell colony formation by DBA/1 bone marrow cells in vitro when compared with serum from conventional Swiss/HaM mice. Sera from germfree preleukemic and leukemic AKR mice exhibited strong colony stimulating activity, indicating that the increased colony stimulating activity previously observed in the serum of conventional leukemic mice is not the consequence of bacterial or fungal infections supervening in leukemic animals with deficient immune responses.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.     

Experimental model for studying the participation of bone marrow cells in antibody formation

Participation of bone marrow cells in the production of IgM antibody forming cells (AFC) in the primary immune response to sheep red blood cells (SRBC) in C57Bl/6 and BDA/2 mice was studied. The animals of this line differed in sensitivity to preoral benz(a)pyren (BP) injection. After BP injection a toxical injury of bone marrow cells was observed for two days in DBA 2 mice but was not marked in C57Bl/6 mice. In the former it was followed by a 10-fold decrease of IgMAFC, while no profound changes were noticed in the immune response of the latter. A new model is offered for the evaluation of bone marrow cell participation. A suggestion is made concerning some connection of immunodepression in the bone marrow with the change of the stem hemopoietic precursor differentiation.     

Resolution and purification of three distinct factors produced by Krebs ascites cells which have differentiation-inducing activity on murine myeloid leukemic cell lines

The use of different myeloid leukemic cell lines (WEHI-3B D+ and M1) and different sources of factors has led to discrepancies concerning the identity of factors capable of inducing differentiation in leukemic cells. We have biochemically fractionated medium conditioned by one such source (Krebs II ascites cells) and assayed fractions for their bone marrow colony-stimulating activity as well as their differentiation-inducing activity for WEHI-3B D+ and M1 cells. This resulted in the resolution of four distinct molecular species with differentiation-inducing activity. One activity was purified to homogeneity and shown by a variety of biochemical, biological, and receptor-binding criteria to be authentic granulocyte colony-stimulating factor (G-CSF). A second activity was identified as granulocyte-macrophage colony-stimulating factor (GM-CSF). Two other activities termed LIF-A and LIF-B (leukemia inhibitory factor) were shown to probably be different glycosylation variants of the same protein and one of these (LIF-A) was purified 12,000-fold to homogeneity. G-CSF induced differentiation in both WEHI-3B D+ and at higher concentrations M1 cells while GM-CSF weakly induced differentiation in WEHI-3B D+ cells. LIF-A had no colony-stimulating activity and induced differentiation in and inhibited the proliferation of only M1 cells. Each factor bound to a unique cell surface receptor with no evidence of direct cross-reactivity.     

Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction?

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.