Studies of passive transfer of cellular resistance, as manifested by refractoriness to necrotization with virulent tubercle bacilli, have shown that immune histiocytes or immune lymphocytes were effective transferring agents; immune polymorphonuclear leucocytes and immune serum as well as comparable cells from normal animals lacked this capacity. Comparisons of immune histiocytes and immune lymphocytes showed that the former cells were more efficient; this was indicated by (a) the smaller numbers of immune histiocytes needed for passive transfer, (b) the longer duration of cellular resistance in recipients given histiocytes than in those given lymphocytes, (c) the greater capacity of histiocytes to effect serial passive transfer, and (d) the ability of histiocytic but not lymphocytic lysates to transfer cellular resistance. Experiments to establish the mechanism of passive transfer of cellular resistance showed that there was no active induction of resistance in recipients through transfer of bacillary antigens contained in immune histiocytes; in fact, the results of serial passive transfers with immune histiocytes suggested an active replication of the "cell resistance factor."
Although various observations (1-5) have failed to relate serum antibody to resistance against tuberculosis, these findings need not preclude a role for humorM factors in an as yet indeterminate and possibly rather complex pattern of resistance. The participation of humoral factors in resistance to tubercle bacilli was indicated by the observation that the sera of animals immunized with the BCG strain of tubercle bacillus contained a substance which protected the monocytes of these immunized animals against the necrotizing action of virulent tubercle bacilli (6). While the protective substance was found in immune and not in normal sera, its activity was seemingly non-specific in nature, for the immune sera of animals immunized with antigens totally unrelated to the tubercle bacillus (Salmonella, Brucdla, ovalbumin) proved equally effective in protecting the monocytes of BCG-immunized animals against virulent tubercle bacilli (7, 8). It was also found that these immune sera protected the monocytes of the BCG-immunized animal but failed to afford similar protection to normal monocytes (6).Studies of cellular resistance showed that it was also not entirely specific, since, in the presence of immune sera, the monocytes of animals immunized with either Mycobacterium or Brucdla proved partially or completely resistant to heterologous as well as homologous infection (8). Despite the seeming lack of specificity suggested by the cross-immunity between the cells of animals immunized with Brucella and Mycobacterium some selectivity of action was demonstrable in cells; thus, while both anti-Brucdla and anti-Salmonella sera contained a substance which protected the cells of BCG-immunized animals against virulent tubercle bacilli, the monocytes of the Brucella-and Salmonellaimmunized animals behaved quite differently in that the cells of the Salmonellaimmunized animal proved completely susceptible to virulent tubercle bacilli (7).
The various cellular components of immune rabbit histiocytes have been analyzed for their ability to induce cellular resistance in normal animals. The results of these investigations have shown that the nuclear and mitochondrial fractions were inactive and that the microsomal and ribosomal fractions were active. The importance of ribonucleic acid in induction of cellular resistance was established by isolation of an active ribosomal RNA and by demonstration of inactivation of this material with ribonuclease but not with deoxyribonuclease or trypsin. The possibility that viable bacilli were present in immune ribosomes was tested; the absence of complement-fixing antibodies and of skin reactivity to tuberculin in animals inoculated with ribosomes was considered as partial evidence of absence of living bacilli.
There are various observations to indicate that under appropriate conditions of in vitro cultivation, attenuation of virulent tubercle bacilli may be achieved.The BCG strain of tubercle bacillus, originally a virulent bacillus of the bovine type, provides a classical example of in v/tro attenuation after prolonged cultivation on a glycerol-potato-bile medium (1). Similar reductions of bacillary virulence have also been accomplished by cultivation of virulent forms at low hydrogen ion concentrations (2, 3) and by growth beneath the surface of fluid media (4-6).Although naturally occurring in ~/vo attenuation of virulent tubercle bacilli has been reported, particularly in association with lupus (7), it has been the general experience of investigators that experimental attenuation of virulent tubercle bacilli by in v/vo methods was unsuccessful; even prolonged growth of mammalian tubercle bacilli in the bodies of cold-blooded animals has not resulted in any significant reduction of virulence (8).As virulent tubercle bacilli which gain access into animal tissues are largely phagocytosed by mononudear cells, it seemed a reasonable assumption that if destruction of bacilli or alteration of bacillary virulence were to occur, a propitious time for this would be during growth of bacilli in these cells. An earlier paper (9) has shown that passage of virulent tubercle bacilli in an immune system (monocytes of BCG-immunized rabbits cultivated in homologous immune serum medium) resulted in decreased capacity of bacilli to induce degeneration of normal monocytes.The present paper reports the decreased capacity of tubercle bacilli to cause death of mice following passage of virulent bacilli in normal systems (normal monocytes maintained in normal serum medium) as well as in immune systems; it also describes certain other changes in the behavior of passaged bacilli. Materials and MettwdsDetailed descriptions of procedures and materials not previously used are presented below; those which were employed in earlier studies (9--11) are briefly described herein.Monocytes.--Normal adult rabbits and rabbits injected intradermaliy with viable BCG
Earlier reports (1, 2) from this laboratory have shown that immunization of animals with the BCG strain of tubercle bacillus resulted in the development of resistant populations of histiocytes in these animals; this form of cellular resistance was manifested by the refractoriness of infected immune histiocytes to necrotization by virulent tubercle bacilli (H37Rv strain). The expression of such resistance was, however, dependent upon the presence of some non-specific factor present in homologous and heterologous immune sera (e.g. anti-BCG, antiovalbumin, anti-Salmonella sera).Further studies (3) have shown that a similar type of cellular resistance was demonstrable with the histiocytes of rabbits immunized with the Rev I strain of Brucella mel/tens/s. There was, moreover, a state of cross-immunity between the Brucella-immune (from animals immunized with Rev I) and the BCG-immune (from animals immunized with BCG) histiocytes such that infection of one or the other of the two histiocytes with either the homologous or heterologous pathogen did not result in destruction of cells by the parasite. An obvious inference which may be drawn from this observation is the probable identity of the mechanism or mechanisms for this type of cellular resistance.Since the above findings have been reported, additional characterization of the tubercle bacillus-histiocyte model system has been achieved (4--6). It is now known that cellular resistance against mycobacteria can be induced in normal animals by injections of immune histiocytes, recipient histiocytes (histiocytes of normal animals which had been injected with resistant cells), and by immune ribosomes and ribosomal RNA (ribonucleic acid). It is also an established fact that serial transfer of resistance in normal animals against mycobacteria is achievable by injection of animals with resistant histiocytes or subfractions (ribosomes) derived therefrom. Moreover, it has been shown that passage of virulent tubercle bacilli in immune histiocytes resulted in attenuation of bacilli for both mice and normal histiocytes (i.e. passaged bacilli lose their inherent ability to necrotize the histiocytes of normal animals).In view of the above findings, it seemed propitious to investigate the identity or lack of identity of the basic mechanisms which underlie cellular immunity
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