L. A. Fonina

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.

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.

Synthesis and Properties of the retro-Analogue of Myelopeptide MP-2

The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70–82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical.

Synthesis and antitumor properties of the myelopeptide MP-1

The bone marrow-derived peptide Phe-Leu-Gly-Phe-Pro-Thr (MP-1) has been synthesized by the classical methods of peptide chemistry in solution, and its antitumor properties have been studied. It has been shown that MP-1 enhances the efficacy of the cytostatic therapy of lympholeukosis P388, increases the latent period of the growth of P388 tumors implanted in irradiated mice, and reduces the recurrence of the breast adenocarcinoma Ca-755 in mice after the surgery.

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.

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.