by Rem PETROV, Academician, Augusta MIKHAILOVA, Dr. Sc. (Biology) and Larissa FONINA, Cand. Sc. (Chemistry), Shemyakin-Ovchinnikov Institute ofBioorganic Chemistry, Russian Academy of Sciences

Peptide regulation is one of the key problems in modem molecular biology, particularly in molecular immunology. The majority of immune system mediators are polypeptides or proteins by nature. They serve as messengers in the signal transfer between various immunocompetent cells and provide for cell cooperation during the development of immune responses.

Regulatory peptides play a crucial role in the functional activity of the central organs of immunity, the thymus (T) and the bone marrow (B). These two organs are responsible for the development and normal functioning of T- and B-systems of immunity. The complex chain of events, including the differentiation and migration of lymphocytes and other related processes, is mediated by an immense variety of regulatory peptide molecules, the cytokins, derived from the thymus and the bone marrow. These events account for the normal performance of the immune and hematopoietic systems.

In contradistinction to the thymus peptides that were detected in the middle of the 1960s and studied closely, the bone marrow peptides were unknown until the middle of the 1970s. They were identified in our research collective and named myelopeptides (MPs).

MPs were first discovered in vitro in experiments on mixed cultures in which antibody-forming cells (AFC) - the lymph node cells of mice immunized with sheep red blood cells (SRBC) - and murine nonimmune bone marrow cells were separated by a millipore membrane (one that does not let the cells across but is permeable for their products). As a result, the number of AFC in the immune lymph node cells suspension increased 2-3-fold compared with the control culture not containing bone marrow cells under the membrane. So it was proved that bone marrow cells produce humoral factors acting upon the level of antibody production of mature AFC.

The fraction possessing MP activity was isolated from the supernatant of 20 h murine (later, porcine) bone marrow cell culture by gel chromatography on Sephadex G-25. This fraction contained substances with a molecular weight of 500-5,000 Da and possessed the following three main types of activity: immunoregulating, cell-differentiating and neurotropic ones.

As demonstrated by further experiments, MPs are produced by bone marrow cells of animals and humans in the course of their vital activity, without antigenic or mutagenic stimulation; they are not species-specific and, being isolated from bone marrow cells of animal species, can be used for immunocorrective purposes in man. All these facts promoted the development of a new immunomodulator, myelopid, which is widely used in Russian clinics for the treatment of many diseases related to immunological or hemopoietic disorders. It effectively prevents postsurgery and posttraumatic complications, providing for the normalization of immune status in these patients; it also has a positive effect on treatment of chronic bronchitis and pneumonia, bacterial infections, and on combined therapy of acute leukemia.

Chemical structure identification was the first essential step in studying the biological characteristics of MPs and the mechanisms of their action, for which purpose these compounds had to be obtained in sufficiently large amounts. It is clear, however, that these biologically active substances are secreted by metabolizing bone marrow cells into the culture medium at very low concentrations. They are present in the supernatant together with other components: amino acids, vitamins, nucleic bases, salts, etc. - which are found in excess there. That is why it was necessary to develop a special method for peptide isolation that could make possible the separation of very small amounts of biologically active material from the huge body of components contained in the culture medium (about 5001).

To isolate individual MPs from the culture medium, we decided to rely on solid-phase extraction. The supernatant was passed through a high-molecular-weight sorbent (the home-made micropore polymer Forsart) that absorbs not

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only MPs but also culture medium components that, in practical terms, cannot be absorbed otherwise. Then polymer-fixed MPs were eluated. These techniques enabled us to separate the biologically active M Ps from nearly all the components of the culture medium in one stage only and to concentrate the solution in a small volume. The solution thus obtained retained all the biological activities proper to the stock supernatant. Further separation of the MP mixture was performed by reversed-phase chromatography; the collected samples were tested for biological activity at each step of the process.

A comparison of their sequences with those of the studied proteins and polypeptides in the data bank showed peptides MP-1 and MP- 2 to be identical to conservative fragments of hemoglobin alfa - and beta -chains, while MP-3 and MP-6 showed no homology with known sequences.

The identified MPs were synthesized, and their biological properties studied more closely.

Immunocorrective activity of MP-1 was tested in various experimental models of immunodeficiency An gamma -irradiation of mice at a dose of 300 ber caused a decrease of antibody production up to 36.8 percent. Inoculation of MP-1 to irradiated mice on days 0,1,2 and 3 after their immunization with SRBC resulted in an increase of antibody production. The maximal effect (up to 80 percent of the normal level) was observed at a dose of 1 x 10 ^-9 g/mouse. Doses of 1 x 10 ^-6 , 1 x 10 ^-7 and 1 x 10 ^-8 g/mouse caused a less pronounced (up to 50-60 percent) but statistically significant stimulation of antibody formation. Doses of 1 x 10 ^-5 and 1 x 10 ^-10 g/mouse did not lead to immunocorrection. But MP-2, at most of the doses used, had no immunocorrective effect.

Immunocorrective effect of MP-1 was also revealed in mice treated with cyclophosphamide (Cy), a compound that inhibits the immune response. The cytostatic was inoculated into a mice at a dose of 200 mg/kg and, 9 days later, the mice were immunized with SRBC. MP-1 or MP-2 were inoculated 4 times to mice on days 0,1,2 and 3 after immunization. At a dose of 1 x 10 ^-6 g/mouse MP-1 recovered the number of AFC in the spleen ofimmunodeficient mice up to the normal level. Yet MP-2 had no corrective effect on antibody production in Cy-treated mice at any dose used.

Besides the model of immunodeficiency induced by irradiation or cytostatic, NZB mice having congenital disorders of the immune system were used in experiments. Administration of MP-1 to these mice changed some immunological parameters due to its ability (as found in special studies) to inhibit the T-suppressor activity in the productive phase of the immune response. Joint cultivation of mature AFC (mouse immune lymph node cells) and T-suppressors (Con A-activated mouse spleen cells) resulted in a 2-fold decrease of antibody production. The presence of MP-1 in this mixed culture prevented the inhibitory effect of T-suppressors on the number of AFC. This attests to the ability of MP-1 to influence the regulatory subsets of T-lymphocytes (T-helpers or T-suppressors).

Using an FITC-labeled peptide, the binding of MP-1 to specific receptors on target cells was examined. There were 18 percent FITC-positive cells in the mouse spleen cell suspension. The addition of unlabeled MP-1 to the incubation mixture in excessive amounts caused a decrease in the number of FITC-positive cells due to the substitution of FITC-labeled MP-1 by unlabeled MP-1. The binding of MP-1 to target cells was specific, because the addition of another unlabeled hexapeptide (MP-2) did not result in the substitution of the FITC-labeled MP-1 at binding sites. Using FITC-labeled monoclonal antibodies to specific markers of T- helpers and T-suppressors as well as the labeled MP-1, we established that T-helpers were the target cell for MP-1: it binds to T-helpers and normalizes the helper/suppressor balance in immunodeficient mice.

So, MP-1 is an endogenous immunoregulatory peptide which realizes its immunocorrective effect due to interference in helper/suppressor interaction. MP-1 is capable of binding to specific receptors on the surface of T-helpers. Therefore it

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restores the balance of regulatory T-cells-their imbalance is the cause of many immunodeficiencies.

The antitumor activity of MP-2 was first revealed by us in the model of recovery of the human T-lymphocyte proliferative response to the mitogen, say, phytohemagglutinin (PHA), inhibited by tumor toxic products (T-lymphocytes play a crucial role in the immune response to tumors). It is known that conditioned media from HL-60 myeloblastic leukemia cells (HL-60 CM) inhibit the PHA-induced proliferation of human peripheral blood T- lymphocytes that is characteristic of T-lymphocyte functional activity. The addition of HL-60 CM to PHA-stimulated lymphocytes resulted in a 2-fold suppression of cell proliferative responses. MP- 2 recovered the T-lymphocyte response in a dose-dependent manner (up to 95 percent of the norm) at a concentration of 100 mkg/ml.

Flow cytometry analysis showed that human T-lymphocytes exposed to HL CM had a decreased proportion of T-helpers (CD ⁺ ₄ cells). At the same time, there were no changes in the CD ⁺ ₈ antigen expression (T- suppressors). The addition of MP-2 to these "suppressed" T-cells completely restored the CD ⁺ ₄ T-lymphocyte phenotype damaged by leukemia cells.

MP-2 ability to restore T-lymphocyte functional activity inhibited by tumor cells is apparently the basis for the antitumor effect of this peptide in tumor-bearing mice. MP-2, inoculated on the third day after tumor transplantation, inhibited the tumor growth as much as 70-80 percent. This effect was demonstrated on various tumor types: lym-pholeukemia P-338, mammalian adenocarcinoma Ca- 755, sarcoma S-180, melanoma B-16, etc.

Consequently, MP-2 is apparently an endogenous immunoregulatory peptide participating in anticancerogenesis processes in the living organism. It protects the immune system from tumor toxins and thus holds promise for application in antitumor prophylaxis and therapy.

MP-3 stimulates macrophage phagocytosis, i.e. a process whereby macrophages attack and capture foreign particles. This effect was clearly demonstrated on infected mice.

MP-3 stimulates phagocytosis in a dose-dependent fashion. The maximal stimulation of phagocytosis takes place when MP-3 is administered at doses of 10 ^-7 -10 ^-8 g/ml and reaches 250 percent. We obtained these data in model experiments in vitro: macrophages from the murine peritoneal cavity captured sheep red blood cells present in the same culture medium. The degree of phagocytosis could be estimated by an "oxygen explosions" that accompanied the process.

In another experiment, MP-3 was inoculated to mice at doses 0.5 x 10 ^-4 g/mouse (group 1) and 1 x 10 ^-6 g/mouse (group 2). Then, 24 h later, these mice were infected with various doses of Salmonella typhimurium 415 (10 ² , 10 ³ , 10 ⁴ or 10 ⁵ bacterial cells/mouse). The mice of the control group were inoculated with saline solution. While the death-rate of the animals in the control group (10 ⁴ and 10 ⁵ bacterial cells/mouse) was 100 percent, the survival in the groups treated with MP-3 was as high as 70-90 percent. It may well be that this peptide protects the animals from bacterial infection due to its ability to stimulate macrophage phagocytosis and thus can be used in the future as medication in antibacterial therapy.

Initially the HL-60 cell line was obtained from bone marrow cells of a patient afflicted with acute myeloid leukemia. These cancer cells, characterized by low cytodifferentiation, are intensively proliferating myeloblasts which can differentiate to the granuloid or monocyte pathway only in the presence of appropriate differentiating agents. The peptide MP-4 proved to be one such agent.

The human myeloid HL-60 line was maintained in a standard culture medium. After 3 days of cultivation, the cells were washed and reincubated in a fresh culture medium. In another three days, each culture was labeled with ³ H-thymidine and ¹⁴ C-glycine. The cells were harvested on the 6th day of cultivation and their DNA ( ³ H) and protein ( ¹⁴ C) radioactivity was measured. The mean counts per min (cpm) in the triplicate culture were analyzed. As a matter of fact, a simultaneous decrease of chromosomal DNA synthesis (cell proliferation) and an increase of total protein synthesis (cell maturation) are characteristic of the cell differentiation process. Well, MP-4 was found to induce the differentiation process in HL- 60 cells. Its effect on blastoid HL-60 cells is dose-dependent. The optimal effect was observed at doses of 0.1-5.0 mg/ml.

A morphological analysis of HL-60 cells treated with MP-4 confirmed these results. There were about 60 percent mature forms (monocytes and macrophages) among blastoid cells.

Subsequently the differentiating effect of MP-4 was compared with that of the known differentiation factors: phorbol myristate acetate and the T-lymphocyte a maturation inducer. As an endogenous regulatory peptide playing a significant part in cell differentiation, MP-4, no doubt, promised to be good for treatment of various leukemias.

The study of biological activities of myelopeptides MP-5 and MP-6 is in progress now.

Thus, various activities of isolated MPs, their action, both in vitro and in vivo, the dose dependence of MP effects, their selective action on definite target cells as well as their binding to specific receptors on target cells - all that testifies to MPs being a new class of regulatory peptides that play a major role in the vital activity of the organism.

The MP problem is also important for clinical immunology As mentioned above, the medication Myelopid based on MP mixture is being effectively used in Russia for correction of immunological and hemopoietic disorders. A combination of individual, structurally determined MPs, responsible for definite Myelopid effects, opens up a prospect of obtaining an M P mixture suitable for a concrete patient depending on his immune status.

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