AN EXPERIMENTAL STUDY OF THE HISTOGENESIS OF THE MILIARY TUBERCLE IN VITALLY STAINED RABBITS
Authors: Herbert M. Evans, Fred B. Bowman, M. C. Winternitz
Journal: The Journal of Experimental Medicine (1914)

In our study of the histogenesis of the miliary tubercle developing inside the liver lobule in animals that have been stained vitally while inoculated with bovine tuberculosis, the controls enable us to recognize the manner in which the vital stain affects the liver. There is therefore no possibility of confusing the effects due to the organism with the effects due to the dye. It is, however, of interest to note that the effects are closely related. The vital stain alone is able to produce gradually some of the same changes that occur with far greater rapidity in experimental tuberculosis. Although in a few hours the Kupffer cells of tuberculous animals begin to react to the disease, in the case of normal animals stained vitally they do not do this until after the third or fourth dose of successive daily injections. After many days, nevertheless, the vital stain alone produces enlargement, proliferation, and separation of Kupffer cells so that these are converted into large free phagocytes which may possess one or several nuclei. These are the gigantic macrophages of chronically stained animals. In all our experiments we have used only acutely stained animals, so that the effects of the dye itself are never sufficient to produce the changes. In fact there is no evidence that the dye accentuates the changes appreciably during the time involved in the experiment. The dye, however, shows us the type of the cells entering into the tuberculous granuloma, for when fed to the body fluids in abundance trypan blue finds its way into all cells capable of receiving it. The vital stain is, as it were, a physiological test for the cells. Whatever the fundamental nature of the vital stain produced by trypan blue and the benzidine dyes may be, it is important that this reaction does not occur to any appreciable extent with mononuclear blood cells, and that it does occur emphatically in the case of the hepatic endothelium. By means of this vital test, then, the following phenomena occur when suspensions of tubercle bacilli are let into the portal blood stream. The organisms, swept on by the blood stream, finally lodge in the terminal branches of the portal vein, where they plug the vessels and continue to multiply. They injure the vessel wall and cause around them an exudative inflammatory process, and finally lead to the formation of tubercles situated not only in these areas but also within the liver lobule. The injury to the vessel wall is manifested in the early stages by the presence of vitally stained areas in its structure. The bacteria at the end of half an hour are found to be extracellular in clumps in the larger vessels, but already to some extent in the bodies of vitally stained Kupffer cells throughout the liver. Exudative inflammation manifests itself by the presence of a transitory accumulation of polynuclear leucocytes about the bacterial clumps, which may be seen as early as half an hour after the inoculation. They continue to be present in the larger cell clumps of the periportal areas for many days, but they are rapidly replaced by other cells, mononuclear in type, so that within a day the histological appearance of the portal plug has changed radically. The mononuclear cell thus entering most actively into the reaction is endothelial and not hematogenous in origin, the vital stain enabling us to make a clear distinction. This fact, evident in the portal plugs, is decisively shown in the case of tubercles developing within the liver lobule. Such tubercles probably result from the localization of individual organisms within the Kupffer cells, for the initial stages of such a probable cycle have been found by us. They consist of the occurrence of mitoses in certain Kupffer cells where the Ziehl-Nielson method shows a bacillus or several bacilli to have been phagocytized (figure 4). Rapid growth of the infected cell now takes place, and at thirty-six hours the multinucleated giant cell produced is largely separated from the other endothelium of the vessel wall. Many bacilli exist within the protoplasm of these cells (figure 5), which are especially distinguishable by their intense reaction to the vital stain. They have received trypan blue to such an excess that low power views of liver sections at the thirty-six hour stage show these cells as deep blue spots (figure 8). The origin of the giant cell from the Kupffer cell is evident not only from the above sequence and from the elective stain, but also from the fact that even when fully formed, protoplasmic strands still join it to its mother tissue,-the normal endothelium of the vessel. The strands entangle other cells in their meshes, especially mononuclear blood cells, one of which, of the polyblastic type, has homogeneous protoplasm and is not infrequently encountered in mitosis. These cells are unquestionably of importance in the lesion of tuberculosis. We have seen them abundantly in the capillaries soon after the inoculation and they also occur singly or in nests between the Kupffer cells and liver columns. They are, as a rule, free from the vital dye. They continue to be concerned in the further growth of the tubercle and with the connective tissue cells make the structure of older tubercles relatively complex. On the other hand, little complexity occurs in the structure of the young intralobular masses. The miliary tubercle formed at the end of thirty-six hours is composed of a giant cell, surrounded by epithelioid cells and by blood cells of the above polyblastic type. The giant cell and its so called epithelioid cells are electively stained and are exclusively derived from the hepatic endothelium.