In the present condition of the technique of cultivation of tissues, the only possible way of studying leucocytic secretions was to grow colonies of leucocytes in a medium of known properties and to examine the modifications of these properties under the influence of the living cells. The method was far from perfect, because the secretions were mixed with serum and accumulated for 48 hours in a medium where they probably underwent partial destruction. But an approximate idea of certain of the qualities of the secretions, although not of their quantity, could be derived from the experiments. In the fluids extracted from the cultures, we attempted to detect the presence of the leucocytic secretions through their physiological effects on homologous and foreign cells. Two kinds of substances were sought, those which act on homologous cells, and those which destroy foreign erythrocytes.
The secretion by leucocytes of substances necessary to the nutrition of other cells was considered as probable long ago. Renaut thought that the main function of the white blood corpuscles was to bring to the fixed cells of the tissues the food material which they need. While the existence of physiological relations between leucocytes and tissue cells could be considered as almost certain, their nature had remained practically unknown. It was probable, however, that the substances secreted by leucocytes were analogous to the growth-activating and unstable substances which are found in embryonic tissues, leucocytes, and certain adult tissues. When connective tissue was aseptically inflamed, or when an aseptic peritoneal exudate contained many leucocytes, aqueous extracts of both connective tissue and peritoneal exudate were found to have acquired the power of stimulating cell proliferation. These experiments showed that leucocytes could bring to the tissues some activating substances. But it remained to be ascertained whether leucocytes, while they are alive, could secrete similar substances either spontaneously or under the stimulus of a foreign factor.
Leucocytes are supposed to be, as is well know, the origin of the substances which protect the organism against infection. Although the problem of the origin of alexin and antibodies has been investigated by many experimenters, it is not yet completely solved. It was of interest, therefore, to ascertain whether leucocytic secretions could increase the natural hemolytic effect of hen serum on sheep or rabbit erythrocytes, and whether these secretions would become more active under the influence of a foreign protein. The substances which destroy foreign cells are not necessarily different from those which act on homologous cells. The word substance is used for simplicity of description and may be taken as meaning only a given property of an unknown substrate.
A comparison was made of certain properties of sera extracted after 48 hours incubation from media containing leucocytes and from media containing no leucocytes. The serum from the leucocytic cultures was always found to be more favorable to the growth of homologous fibroblasts than the serum from the culture media incubated without leucocytes. The natural hemolytic power of the serum on sheep erythrocytes was found to be increased in about SO per cent of the experiments.
In other experiments, we found that when two culture media free of cells were placed, one in an incubator at +38°C. and the other in a refrigerator at +5°C. for 48 hours, the serum from the incubated medium partly lost its hemolytic action on sheep or rabbit erythrocytes, while that from the refrigerated medium remained normal; at the same time, the inhibiting action of the incubated medium on homologous fibroblasts had increased very much. This effect of incubation indicates that certain unstable constituents of serum are destroyed by heat. Then the changes found in the properties of the serum from cultures of leucocytes are due to the fraction of the activating substances which has not been destroyed by incubation at 38°C. A quantitative study of the secretions is, therefore, impossible with the present technique, which can furnish only qualitative indications about the substances set free by the leucocytes.
We have ascertained also whether a medium containing leucocytes and kept in the refrigerator undergoes any change under the influence of the cells while they are in a condition of latent life. Gabritschewski dishes with and without leucocytes were placed in a refrigerator at a temperature of about +5°C. After 48 hours, the hemolytic power on sheep erythrocytes of the serum from the leucocytic cultures had increased slightly and its inhibiting action on the growth of homologous fibroblasts had decreased. Then certain substances favorable to the growth of homologous cells and toxic for heterologous cells were diffused by the leucocytes into their medium. But the action of these substances was weaker than in the case of the cultures kept in the incubator. This experiment showed that leucocytes under certain conditions diffuse alexin or natural hemolysins which originate from them at the same time as the substances which activate homologous cells. In other experiments, although leucocytes were frozen at –10°C., treated with distilled water, or extracted with saline solution, they did not yield any hemolysin.
To summarize: Leucocytes, cultivated in plasma, always secreted substances which increased the rate of growth of homologous cells. Less frequently, they set free substances which hemolyzed foreign erythrocytes.
The growth-promoting substances are analogous to those contained in embryonic tissues, and probably represent some of the foodstuffs brought to fixed tissue cells by leucocytes. They may possess the function of rejuvenating cells which have ceased to multiply when the cicatrization of a wound or the repair of a fracture requires a resumption of tissue activity. According to this hypothesis, the leucocytes brought to the surface of a wound by the process of inflammation would not only oppose bacterial invasion, but also bring to the tissues the material necessary to cell multiplication. It seems that in some cases regeneration is started by substances brought to the tissues by other cells. Loeb thinks that in Tubularia, when endodermic cells gather at the end where a new polyp is about to be formed, the substances given off by these cells are responsible for polyp formation.6 There may be an analogy between this phenomenon and the secretion by leucocytes of growth-activating substances at the surface of a wound.
If we assume that leucocytes in vivo set free their secretions in the blood stream, certain variations of the growth-inhibiting action of normal serum can be better understood. The rate of proliferation of homologous fibroblasts is much slower in the serum of an old chicken than in that of a young one. When the serum is heated at 56° and 70°C. for ½ hour, it becomes still more inhibiting. A substance favorable to cell activity has disappeared. It is therefore permissible to suppose that the growth-inhibiting power of serum and its variations are due to the antagonistic action of two substances, one growth-promoting and thermolabile, and the other growth-inhibiting and thermostable, the activating substance being always weaker in its effect than the inhibiting one. We know that activating substances can be extracted from embryonic tissue, from muscle and gland tissues, and from leucocytes of the adult animal, and that they are thermolabile and very unstable. Leucocytic secretions seem to have some of the properties of leucocytic extracts. It is probable that the activating substances which disappear from the heated serum are secreted by leucocytes and other cells. An increase of these secretions, then, would diminish the inhibiting action of serum on homologous fibroblasts. On the contrary, a decrease of the secretions in the serum would increase its inhibiting effect on homologous cells. The strong inhibiting action of serum in old age would be due partly to a reduction in the amount and activity of the substances secreted by leucocytes and tissue cells in the humors of the organism.
Leucocytes also secreted in vitro substances which were toxic for foreign cells. Although the results were not constant, the serum appeared to become slightly more hemolytic for sheep or rabbit erythrocytes, under the influence of the leucocytes. The hemolysis of rabbit corpuscles by hen serum is due, according to Hyde,9 to a complex sensitizer alexin, and not merely to alexin, as Bordet thought.
When a foreign protein was added to the culture medium, the leucocytic secretions increased, as was shown by the action on homologous fibroblasts of sera taken from cultures of leucocytes with and without casein. The presence in the medium of the cultures of leucocytes of only 0.1 per 1,000 casein did not markedly modify the action of their serum on the proliferation of fibroblasts. When the concentration of casein in the leucocyte cultures reached 1 per 1,000, the growth of chicken fibroblasts in the serum extracted from the Gabritschewski dishes became more rapid. But there was no parallel increase of the hemolytic action of the serum upon sheep erythrocytes.
We found that chicken serum containing 0.1 per 1,000 casein was barely toxic for homologous fibroblasts, while it became markedly inhibiting when the casein concentration reached 1 per 1,000. Probably, there is a relation between the toxicity of the medium, the increase of leucocytic secretions, and the time of the increase. The change brought about by casein in the equilibrium of the system composed of the cells and their medium determines the secretion by the leucocytes of substances which increase the activity of homologous cells and oppose the inhibiting effect of the foreign proteins. This reaction of the leucocytes is immediate, and may represent the first defense of the organism against a factor which disturbs its equilibrium. Possibly it differs from the specific cell reaction which leads to the production of antibodies. It is known that antibodies develop more slowly. Hemolysins were detected in cultures of bone marrow 4 days after the addition of antigen. The immunization of fibroblasts against foreign proteins has been shown by Fischer to begin after 4 days. If leucocytes behave in the organism as they do in vitro, we may assume that before the appearance of antibodies, they respond to the presence of an antigen by setting free growth-promoting substances and possibly alexin. This immediate reaction of the leucocytes against a disturbing factor, and the resulting production of substances which increase the activity of homologous cells, might be partly responsible for the results observed in the treatment of certain diseases by the injection of foreign proteins.
It may be concluded that, under the conditions of the experiments:
1. The serum obtained from cultures of leucocytes is less inhibiting for homologous fibroblasts than the serum from media without leucocytes. In some experiments, its hemolytic action on sheep or rabbit erythrocytes is also increased.
2. The addition of casein to leucocytic cultures brings about a decrease in the inhibiting effect of the serum on homologous fibroblasts.
3. The increase in the activity of homologous fibroblasts in serum obtained from leucocytic cultures is probably due to growth-promoting substances secreted by the leucocytes. The presence of a foreign protein under certain conditions determines a more abundant leucocytic secretion.