Roles of adroxazine, a new heterocyclic hormone of the adrenals, and leucogenenol, a thymothyroid hormone, in the formation of blood cells.

Treatment of normal rats with the thymothyroid hormone leucogenenol accelerates the rate at which the 'functional' cells, neutrophils, red blood cells and lymphocytes, develop in the bone marrow from their corresponding committed precursor cells. Following the initial treatment of normal rats with leucogenenol, there is a temporary elevation in the bone marrow of the relative concentrations of myeloblasts, rubriblasts and lymphoblasts. It has now been found that bilaterally adrenalectomized rats have a different response from normal rats to treatment with leucogenenol. Although treatment of bilaterally adrenalectomized rats with leucogenenol accelerates the rate of development of their functional cells, the initial injection of leucogenenol is followed by a temporary decrease in the relative concentrations of myeloblasts, rubriblasts and lymphoblasts in their bone marrow. Additionally, adrenalectomized rats treated concurrently with tritiated thymidine and leucogenenol show a significant lower percentage of labeled cells in their bone marrow than do correspondingly treated normal rats. These results indicate that adrenalectomized rats have a lower than normal concentration of stem cells in their bone marrow that can be committed to become functional cells. Treatment with adroxazine, a new recently isolated heterocyclic hormone of the adrenals, causes adrenalectomized rats to respond as normal rats to the injection of leucogenenol. There is a temporary elevation of myeloblasts, rubriblasts and lymphoblasts in their bone marrow as well as a normal increase in the percentage of cells that are labeled following concurrent treatment with tritiated thymidine and leucogenenol. It may be concluded that treatment with adroxazine increases the rate of replication of uncommitted bone marrow stem cells while treatment with leucogenenol increases the rate at which committed cells develop into their respective functional cells. A scheme is presented to show the suggested roles that leucogenenol and adroxazine play in regulating the formation of blood cells.

Adroxazine, a hormone of the adrenals that stimulates the rate of replication of bone marrow stem cells.

Treatment of normal animals with the thymothyroid hormone, leucogenenol, increases the rate at which appropriate committed precursor cells of the bone marrow develop sequentially into their corresponding functional cells: neutrophils, lymphocytes and red blood cells. Hence following treatment with leucogenenol, at a time that is dependent on the quantity of leucogenenol injected, there is a temporary increase in the bone marrow of the relative concentrations of early forms of committed cells such as myeloblasts. However, following treatment of bilaterally adrenalectomized rats with leucogenenol the early forms of committed cells in the bone marrow, such as myeloblasts, instead of showing a temporary relative increase show a temporary significant decrease in concentration. A new compound, adroxazine, C34H65NO2, was isolated from the methanol extract of bovine adrenal tissue. This compound, on injection into bilaterally adrenalectomized rats, causes leucogenenol to have the same effect on the development of their bone marrow cells as it does in normal rats. Adroxazine is present in the cortex of the adrenals and in blood serum. It is suggested that the population of primitive replicating cells is controlled by the concentration of adroxazine in the serum.

Association of leucogenenol, a thymothyroid hormone, with carrier proteins in the thymus.

Leucogenenol a heterocyclic enolic thymothyroid hormone (MW 383) whose concentration in the serum regulates the rate at which already committed cells of the bone marrow develop into functional cells, was found to be associated in the thymus with a carrier protein. The carrier protein for leucogenenol is not precipitated by heating to 80 degrees but following this treatment leucogenenol is precipitated in association with proteins precipitated by acetone and then by saturated ammonium sulfate. On chromatography on Sephacryl G-200 it was found that leucogenenol was associated with proteins of MW approximately 38,000. Leucogenenol is not eluted from the chromatographic column if it is not associated with its carrier proteins. It is suggested that other hormones such as those associated with the reproductive cycle or compounds that result from tissue damage induce the liberation of leucogenenol from its carrier protein in the thymus to the circulation where it is associated as previously described, with a protein of approximately MW 300,000.

Effect of leucogenenol, a thymothyroid hormone, on the growth of immature and neonatally thymectomized rats.

The reports of early investigators that growth is delayed by thymectomy of immature animals have been confirmed. Although growth is delayed by thymectomy, thymectomized animals approach asymptotically with age the same final weight as corresponding intact animals. Treatment with leucogenenol, a thymothyroid hormone, accelerates the rate of growth of immature neonatally thymectomized rats to that of normal rats. However, treatment with leucogenenol does not increase the rate of growth of normal rats. Treatment with leucogenenol does not change levels of growth hormone (GH) or thyroxine (T4) in the serum of either thymectomized or intact immature and adult rats. Neither is the depression in levels of serum leucogenenol that follows thymectomy associated with a change in serum levels of GH or T4. Thus it is apparent that levels of serum leucogenenol do not affect the rate of growth of immature animals by increasing serum levels of GH or T4. By analogy with the finding that treatment with leucogenenol increases the rate at which committed cells of the bone marrow and cells involved in the immune response develop into functional cells, it is suggested that the levels of serum leucogenenol are one of the factors that determine the rate at which types of body cells that make up bone and other body tissues develop from committed precursors.

Effect of leucogenenol, a thymothyroid hormone, on the enzymes and several other constituents of the serum.

It has been found that rats treated with the thymothyroid hormone, leucogenenol, have significantly elevated levels of alkaline phosphatase and lactate dehydrogenase in their serum 8 hrs. later. These levels remain elevated for 24 hrs. and 12 hrs., respectively. The concentrations of creatine phosphokinase and aspartate aminotransferase as well as total protein, albumin, calcium, inorganic phosphorus, glucose, urea nitrogen, uric acid and creatinine in the serum are not affected by treatment with leucogenenol. These results are in agreement with the previously reported findings that treatment with leucogenenol increases the rate of development of blood cells of the bone marrow, increases the rate of recovery from immunosuppression and enhances the immune response.

Effect of leucogenenol, a thymothyroid hormone, on allograft rejection and hemolysin formation.

Recent investigations in this laboratory have established that daily treatment with leucogenenol, an enolic heterocyclic hormone synthesized by the thymus and/or thyroid, induces neonatally thymectomized mice that have accepted a skin allograft to reject this allograft in 7 to 14 days. Also, daily treatment with leucogenenol causes neonatally thymectomized mice to respond to challenge with sheep erythrocytes with the formation of normal titers of hemolysin. These results demonstrate that the hormone leucogenenol induces the development of those cells, absent in neonatally thymectomized mice, that are necessary for a normal immune response.

Effect of tumors on the concentration of leucogenenol in the serum of mice.

It was found that inoculation of several strains of mice with several types of tumor cells resulted, within 24 hr, in a significant decrease in the serum leucogenenol levels of the mice. Serum leucogenenol levels of the mice inoculated with tumors that are rejected become normal or temporarily above normal at approximately the time the tumor is observed to be rejected. Contrariwise, serum leucogenenol levels of mice inoculated with tumors that are not rejected remain at significantly lower than normal levels during the life of the mice. Unlike tumors, skin allografts increase serum leucogenenol levels. When tumors are rejected because of the previous immunization of the mice, serum leucogenenol levels become normal at approximately the time the tumor is observed to be rejected. Excision of the tumor after 1 week of growth, with the consequent recovery of the mice, is accompanied by a recovery of normal serum leucogenenol levels. Also, it was found that injection of mice with a cell-free 0.9% NaCl solution extract of a tumor results in a temporary decrease in serum leucogenenol levels comparable to that observed with the inoculation of a viable tumor which lasts from 24 to 96 hr. It is suggested that the suppression of serum leucogenenol levels is one of the factors responsible for the immunosuppression associated with a growing tumor.

Effect of Leucogenenol on recovery from immunosuppressin by anti-lymphocyte globulins.

Daily treatment of rats with the thymothyroid hormone leucogenenol after their immunosuppression with anti-lymphocyte globulins causes the rats to regain their immunocompetency after 7 days of treatment, in contrast to well over a month, which is required for untreated controls. Immunocompetency was mesured by the ability of the rat to respond to the injection of sheep erythrocytes with the formation of normal concentrations of antibody-forming cells in their lymphoid tissues and normal titers of hemolysins in their serum. These results indicate that leucogenenol increases the rate of development of T cells, as well as B cells, from committed precursors.

Leucogenenol a thymothyroid hormone.

Thymectomy and, to a lesser extent, thyroidectomy cause a significant decrease in the serum leucogenenol levels of rats. Daily injections of neonatally thymectomized mice with leucogenenol causes the mice to maintain a normal level of neutrophils and lymphocytes in their circulation. The thymus and thyroid are the only tissues in rats and mice that afford radioactive leucogenenol when incubated in a medium containing radioactive D-glucose. It is concluded that the thymus and possibly the thyroid are responsible for the normal biosynthesis of leucogenenol in animals.

Leukocyte inhibitory factor (LIF) production from lymphocytes stimulated by leucogenenol.

It has been demonstrated that human peripheral lymphocytes incubated in a tissue culture medium containing 0.04, 0.1, or 0.15 microgram/ml of leucogenenol form or release a factor that inhibits the migration of human peripheral PMN leukocyte. The factor is chromatographed of Sephadex G-100 and migrates on electrophoresis as an albumin, thus suggesting that it has the electric charge of an albumin. The factor is stable to neuraminidase and to heating at 56 degrees C for 30 min, but it is inactivated by heating at 80 degrees C for 60 min. Its physical and biological properties suggest that the factor is identical to the LIF reported by Rocklin.

Effect of skin or muscle incisions or the loss of blood on serum leucogenenol level.

It has been found that damage to a tissue of a rabbit or a rat, such as results from a skin incision or an incision through the skin and muscles into the abdominal cavity, is followed 24 hr later by a significant increase in the concentration of leucogenenol in the animal's serum. Likewise, loss of approximately one-quarter to one-half of the blood in the circulation of rabbits or rats causes an increase 24 hr later in the animals' serum leucogenenol concentration.

Association of leucogenenol with specific proteins in serum.

Determination of the concentration of leucogenenol in the IgM, IgG, albumin, and low molecular weight protein fractions of serum demonstrated that leucogenenol is found only in protein fractions of molecular weights in the range of IgM and IgG. Determination of the distribution of leucogenenol in chromatographically separated serum proteins showed that leucogenenol has a maximum concentration in the proteins intermediate between those with the molecular weights of IgM and IgG. Comparison of the ratio of effluent to void volumes with the ratios of known proteins suggest that the associated or carrier protein of leucogenenol has a molecular weight of approximately 3 x 10-5. When additional leucogenenol is added to serum it is found in the same protein fractions as the leucogenenol normally present, indicating that there is excess carrier protein for leucogenenol in the serum.