Augusta A. Mikhailova

BONE MARROW IMMUNOREGULATORY PEPTIDES (MYELOPEPTIDES) : ISOLATION, STRUCTURE, AND FUNCTIONAL ACTIVITY

Myelopeptides (MPs) are bioregulatory mediators of bone marrow origin. Several individual MPs have been isolated from the supernatant of porcine bone marrow cell culture by successive solid phase extraction and reversed-phase high performance liquid chromatography. Two of them, MP-1 (Phe-Leu-Gly-Phe-Pro-Thr) and MP-2 (Leu-Val-Val-Tyr-Pro-Trp), were synthesized and their biological activities were comprehensively studied. Both hexapeptides display pronounced immunoregulatory activity but their final effects as well as mechanisms of action are different. Peptides MP-1 and MP-2 are identical to conservative fragments 33-38 alpha- and 31-36 beta-chains of hemoglobin, respectively. The sequences of other isolated MPs have no homology with any functional protein. The role of MPs in bioregulatory processes in vivo is discussed.

Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture

Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture. It was synthesized and its immunoregulatory properties were studied. MP-1 caused a 1.5-2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells. It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production. The inoculation of MP-1 (1 x 10(-9) g/mouse) to mice two weeks after their gamma-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 +/- 15.5% as compared to that in the irradiated control 37.6 +/- 12.0%. Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells. It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

Myelopeptides: Bone marrow regulatory mediators

Bone marrow cells of various animal species and men produce a group of bioregulatory peptides called myelopeptides (MPs). A highly purified MP fraction and some individual molecules have been isolated from the supernatant of porcine bone marrow cell cultures by reverse phase chromatography.MPs have a wide spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They evoke 2–5-fold stimulation of antibody production to various antigens. They correct some immune defects in MRL/lpr mice with spontaneous autoimmune disorders that results in 2-fold prolongation of the life span of these mice. MPs influence the differentiation of bone marrow and peripheral blood cells derived from healthy and leukemic donors. They induce terminal differentiation in the leukemic human HL-60 cell line. MPs also show an effect on pain sensitivity.A new immunocorrective drug Myelopid has been developed on the basis of MP mixtures. This drug is effectively used in Russia both in medicine and veterinary practice for prophylaxis and treatment of diseases accompanied by immunodeficiency.Two individual MPs were isolated and identified: Phe-Leu-Gly-Phe-Pro-Thr (MP-1) and Leu-Val-Val-Tyr-Pro-Trp (MP-2). MP-1 displays immunoregulatory activity; MP-2 abolishes the inhibitory effect of leukemic cells on T-lymphocyte functional activity.MPs seem to provide not only immunoregulation but also to participate in complex interactions between different systems in the organism.

The bone marrow peptide (myelopeptide-2) abolishes induced by human leukemia HL-60 cell suppression of T lymphocytes

Myelopeptide-2 (MP-2) Leu-Val-Val-Tyr-Pro-Trp originally isolated from the supernatant of porcine bone marrow cell culture was examined for its capacity to restore the mitogen responsiveness of human T lymphocytes inhibited by conditioned media from HL-60 leukemia cells (HL-60 CM). MP-2 added to phytohemagglutinin (PHA)-stimulated T lymphocytes together with HL-60 CM abolished the suppression of T-lymphocyte proliferative response in a dose-dependent manner. Another bone marrow hexapeptide Phe-Leu-Gly-Phe-Pro-Thr, MP-1, did not display this action in that experimental system. MP-2 was also effective being added after T-lymphocyte exposure to HL-60 CM which suggests its recovery but not protective effect on T-lymphocytes treated with tumor cell products. Flow cytometry analysis revealed HL-60 CM influence on the expression of CD3 and CD4 T-cell surface antigens. It decreased the content of CD3- and CD4-positive cells and induced the appearance of T lymphocytes with reduced density of CD3 and CD4 antigens. MP-2 was able to restore the T-cell phenotype altered by HL-60 CM. MP-2 seems to be promising in anti-tumor therapy.

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.

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.

Myelopeptide-2 recovers interleukin-2 synthesis and interleukin-2 receptor expression in human T lymphocytes depressed by tumor products or measles virus.

Myelopeptide-2 (MP-2; Leu-Val-Val-Tyr-Pro-Trp), originally isolated from the supernatant of porcine bone marrow cell culture, is able to restore the mitogen responsiveness of human T lymphocytes inhibited by conditioned medium from HL-60 leukemia cells or measles virus. This effect is based on the ability of MP-2 to recover the reduced interleukin (IL)-2 synthesis and IL-2 receptor (IL-2R) expression in human T lymphocytes treated with these harmful agents. The involvement of other cytokines in MP-2 restoration of the reduced IL-2 synthesis in T lymphocytes is experimentally studied. It is shown that T helper (TH) 1 and TH2 cytokines are acting in close interaction, the character of which depends on the immune status of the T-lymphocyte donors. The data obtained allow one to suggest that the MP-2 involvement in regulatory processes is directed to the maintenance of immune homeostasis. This peptide is perspective to be applied in antitumor and antivirus therapy.

Immunoregulatory effects of two bone-marrow hexapeptides (myelopeptides) in experimental models of immunodeficiency

Myelopeptide 1 (MP-1, Phe-Leu-Gly-Phe-Pro-Thr) and myelopeptide 2 (MP-2, Leu-Val-Val-Tyr-Pro-Trp) are hexapeptides originally isolated from porcine bone-marrow cell culture. MP-1 enhanced the number of antibody-forming cells in NZB mouse spleen cells in vitro and increased the antibody production in Cy-treated (CBAxC57B1/6)F1 mice in vivo. MP-1 increased ConA-induced proliferation of both NZB spleen cells in vitro and spleen cells of Cy-treated mice in vivo. MP-1 restored polyclonal IgG synthesis depressed by PWM in the NZB spleen cell culture. MP-2 also evoked immunoregulatory effects which were less pronounced and realized at higher concentrations than effects of MP-1. Neither MP-1 nor MP-2 affected LPS-induced spleen cell proliferation in both NZB- and Cy-treated (CBAxC57B1/6)F1 mice.

Myelopeptide-2 induces the resistance of T-lymphocytes to tumor suppression.

Myelopeptide-2 (MP-2) is classified with bone-marrow immunoregulatory mediators, which were discovered by Russian scientists [4]. After isolation of MP-2 from the supernatant of a bovine bone-marrow cell culture and determination of its structure (Leu–Val–Val– Tyr–Pro–Trp), MP-2 was synthesized [3]. The studies of the MP-2 biological activity showed that it is able to restore in vitro the proliferative response of T-lymphocytes to phytohemagglutinin (PHA), which was suppressed by a conditioned medium of HL-60 human myeloleucosis cells. The restoration of the proliferative response was accompanied by the restoration of the tumor-injured phenotypes of CD3+ and CD4+ T-cells [10]. On the basis of these data, the effect of MP-2 on the tumor growth has been studied. It was found that MP-2 suppressed the growth of a number of transplantable mouse tumors (subcutaneously transplanted lympholeucosis P-388, breast-gland adenocarcinoma Ca755, and melanoma B-16) in vivo but did not affect the growth of melanoma B-16 transplanted into the thymusless nude mice. In addition, MP-2 had no effect on the tumor-cell growth in vitro [1, 7]. Thus, it was discovered that the immune T-system plays a key role in the MP-2 antitumor effect.

Purification and properties of a 155 kDa bone marrow-derived glycoprotein enhancing the activity of granulocyte-macrophage colony stimulating factor (GM-CSF)

A protein has been purified from pig bone marrow which enhances the activity of exogenously added granulocyte‐macrophage colony stimulating factor (GM‐CSF) on mouse myelopoiEtic colony formation in vitro. The substance alone did not exhibit any colony stimulating activity. The active fraction was isolated from the medium of pig bone marrow suspension cultures during 20–24 h. Purification to homogeneity was then performed by subsequent HPLC and SDS‐PAGE. The GM‐CSF enhancing protein was identified as a 155 kDa glycoprotein.