BCG-treated Mice Research Articles

The role of BCG/PPD-activated macrophages in resistance against systemic candidiasis in mice.

The main conclusions of this study are that BCG/PPD-activated macrophages, in contrast to macrophages from control mice, exhibit an increased PMA-induced production of H2O2, kill about one-third of the phagocytosed Candida albicans, and cause more than 50% inhibition of the intracellular formation of germ tubes by C. albicans. Peritoneal macrophages from mice that were colonized post-natally with C. albicans do not show increased production of H2O2 upon stimulation with PMA and the intracellular outgrowth of germ tubes is inhibited to only a limited degree. These macrophages are capable of killing about 20% of the ingested C. albicans. In vivo, the number of Candida in the kidney, spleen and liver after intravenous injection of Candida albicans is significantly lower in BCG-treated mice than in control mice. Post-natal colonization with C. albicans has only a limited effect on the outgrowth of intravenously injected C. albicans in the spleen and liver but does not influence growth in the kidney. These results indicate that acquired immunity against a systemic Candida infection involves both oxidative and non-oxidative mechanisms of intracellular killing and that these mechanisms may have different effects on the yeast and hyphal forms of C. albicans.

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse: II. Suppression of pathogenesis by macrophage transfer from BCG-vaccinated mice

Our previous reports showed that a single injection of live BCG, one of the biological response modifiers, prevents insulitis and overt diabetes in NOD mice and that the suppression could be due to the generation of some type of suppressor cells in the BCG-treated mice. Furthermore, a more recent study has revealed that macrophages suppressive against a variety of lymphocyte functions can be induced by BCG, which suggests that these macrophages are involved in the suppression of the pathogenesis. To obtain valid evidence for this speculation, the effects of transfer of macrophage and T-cell fractions on the pathogenesis were examined in the present study. Transfer of macrophage-enriched spleen cell fraction harvested from the BCG-treated females to young females abolished the occurrence of spontaneous diabetes up to the age of 25 to 30 weeks. Also, macrophage transfer prevented the progress of insulitis. In contrast, transfer of a T-cellenriched fraction did not suppress insulitis and overt diabetes. From these results, it could be concluded that the suppression of the autoimmune pathogenesis of diabetes by BCG is due to the generation of suppressor macrophages.

Effects of Stimulated or Immunologically Activated Macrophages on the Induction of Immune Responses to Listeria monocytogenes

The influences of peritoneal macrophages induced by proteose peptone, Corynebacterium parvum (C. parvum) or Bacillus Calmette Guérin (BCG) on the initiation and development of immune responses and protection against Listeria monocytogenes infection were studied in mice. Mice treated intraperitoneally (i.p.) with proteose peptone 4 days previously showed much the same level of protection against an intraperitoneal infection with Listeria as untreated mice. Mice treated i.p. with C. parvum 4 days previously, of which peritoneal macrophages had increased abilities for intracellular killing of Listeria and 0 - 2 generation as compared with peptone-elicited macrophages, exhibited an enhanced resistance against the listerial infection. The degree of immune responses, as assessed by delayed footpad reaction (DFR), was rather depressed in these mice because C. parvum-activated macrophages acting as scavenger cells reduced the amount of effective antigenic stimulation. BCG-activated peritoneal macrophages from mice treated i.p. with BCG 14 days previously showed a strong ability for antigen presentation in correlation with increases in the number of la-bearing macrophages and in the level of interleukin 1 (IL 1) production. These mice showed an early appearance of DFR response and a markedly enhanced resistance against the listerial infection. These results suggested that the differences in macrophage activities as scavenger cells, cytokine-secreting cells and antigen presenting cells may account for the differences in the responsiveness against listerial infection in peptone-, C. parvum- and BCG-treated mice.

Role of oxygen-dependent mechanisms in monoclonal-antibody-induced lysis of normal T cells by phagocytes: II.—Murine phagocytes

In the first part of this study, we reported that antibody-dependent cellular cytotoxicity (ADCC) mediated by human polymorphonuclear (PMN) and mononuclear (MN) phagocytes against anti-T-cell monoclonal-antibody-(mAb)-coated T lymphocytes, is mainly dependent upon the generation of reactive oxygen intermediates (ROI) for PMN, whereas MN-mediated ADCC depends on both oxidative and non-oxidative cytolytic events. Using mouse effector cells from various organs and at various maturation or activation states, the present report shows that in this ADCC model against mAb-coated normal T cells, resident spleen (SPC) and peritoneal exudate (PEC) cells selectively bound and developed oxidative responses to these mAb-coated normal target cells but remained ineffective in ADCC. Similar data were obtained with inflammatory recruited (thioglycollate-elicited or Biogel granuloma-induced) macrophages, whereas immunologically activated macrophages (from BCG-treated mice) mediated both strong oxidative responses and potent ADCC reactions. In contrast, and as in the human system, both resident (from bone marrow) and inflammatory PMN phagocytes exerted significant lysis of these mAb-sensitized normal lymphoid cells. These findings, which are similar to those reported in ADCC against tumour target cells, strongly suggest that the nature (normal or tumoral) of the target cell does not influence the lytic

Role of activated macrophages in resistance to systemic candidosis.

To evaluate further the contribution of activated macrophages in resistance, the course of systemic candidosis was assayed in beige and NLM mice that had been previously infected with Mycobacterium bovis (BCG). Four weeks following BCG infection, mice were inoculated intravenously with 1 x 10(4) viable Candida albicans. At various times thereafter, the number of C. albicans colony-forming units in the livers, spleens, and kidneys was determined. The average number of CFU recovered from the kidneys of NLM mice decreased throughout the assay and was comparable in both BCG-treated and control mice. In contrast, the number of CFU cultured from the kidneys of untreated control beige mice progressively increased throughout the assay period. This profile of renal susceptibility was not appreciably altered in BCG-treated beige mice. However, fewer (10- to 100-fold) CFU were cultured from the livers and spleens of BCG-treated beige and NLM mice than from untreated controls. These results support the hypothesis that in the absence of functional polymorphonuclear leukocytes, activated macrophages represent a means to control the proliferation of C. albicans.

Antitumor activity of cyclophosphamide and lipopolysaccharide in tumor-bearing mice pretreated with BCG.

Antitumor effects of cyclophosphamide (CY) and lipopolysaccharide (LPS) were investigated in BCG-treated mice. C3H/He mice and CDF1 mice were injected with BCG and were inoculated subcutaneously with syngeneic mouse hepatoma and mastocytoma P815 respectively. A subsequent injection of LPS caused hemorrhagic necrosis and retarded growth of tumor. When mice were treated with LPS plus suboptimal dose of CY, tumor growth was retarded and survival time was prolonged. The antitumor effects were more remarkable when mice were treated with CY prior to the injection of LPS. Without BCG pretreatment, LPS showed no antitumor activity in mice. Sera from mice treated with BCG plus LPS was cytotoxic for cultured tumor cells. However treatment of mice with CY did not increase the in vitro cytotoxicity. In this experimental condition, CY had no effect on delayed type hypersensitivity when evaluated by the footpad reaction to purified protein derivative (PPD). These results seem to indicate that the antitumor effects of the treatment with CY and LPS in BCG-treated mice are mediated by the reduction of tumor burden by CY and a serum factor induced by LPS.

Requirement of a Thymus Dependent Immune Response for Bcg-mediated Antitumor Activity

AbstractSurgical adjuvant intravesical bacille Calmette-Guerin (BCG) therapy is an effective method of treating superficial transitional cell carcinoma of the bladder. The role of the immune response in the antitumor activity of intravesical BCG is not known. We investigated the requirement of a thymus-dependent immune response for the inhibition of the growth of the intravesically implanted mouse bladder tumor, MBT-2.Intravesical BCG had no antitumor activity when administered to athymic nude mice bearing MBT-2 tumors. In two experiments tumor outgrowth in control and BCG-treated mice was identical Adoptive transfer of BCG sensitized splenocytes (one spleen equivalent per mouse injected intravenously immediately prior to the first BCG treatment) syngeneic to the MBT-2 tumor transferred delayed hypersensitivity reactivity to BCG antigens and restored the antitumor activity of intravesical BCG. In two separate experiments mice receiving splenocytes plus BCG had 0 and 20% tumor outgrowth compared with 100% in control mice (p <.02 and P <.05, respectively). These results demonstrate that the antitumor activity of intravesical BCG therapy requires a thymus-dependent immune response.

Inhibition of murine bladder tumor growth by bacille Calmette-Guerin: Lack of a role of natural killer cells

Intravesical instillation of bacille Calmette Guerin (BCG) currently is considered the most effective treatment for recurrent superficial bladder cancer, but little is known about the mechanism of action. We have adapted a model in which the mouse bladder tumor, MBT-2, is implanted directly into the bladder to examine the mechanism by which BCG inhibits tumor growth. The intravesical administration of BCG inhibited MBT-2 implantation in a dose-dependent manner. Concomitantly, natural killer (NK) activity was augmented in a dose-dependent manner. Conversely, BCG doses which did not augment NK activity lacked antitumor activity. Linear regression analysis showed a significant correlation between the antitumor activity of BCG and modulation of NK activity (correlation coefficient, 0.991). Additional studies were performed in which NK activity was abrogated by administration of anti-asialo-GM 1 serum. NK activity was depressed in spleens and lymph nodes of both normal and BCG-treated mice. Abrogation of NK activity did not alter the efficacy of BCG therapy suggesting that NK cells are not a major contributor to the antitumor activity of BCG.

Influence of Bcg Administration Time on the in-Vivo Migration of Leukocytes

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten microliter of a 5 X 10(6) live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Non-specific migration was thought to occur and the peak value of leukocytes of 22.8 +/- 6.1 cells/10,000 micron 2 was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.

Antitumor activity of lipopolysaccharide in tumor-bearing mice pretreated with BCG.

The antitumor activity of lipopolysaccharide (LPS) was investigated in BCG-treated mice. C3H/He mice and CDF1 mice were injected with BCG and then were inoculated with syngeneic mouse hepatoma MH134 and mastocytoma P815 respectively. Hemorrhagic necrosis and retarded growth of tumor were observed after an intravenous (i.v.) injection of LPS, when tumor cells had been inoculated subcutaneously (s.c.). However an intraperitoneal (i.p.) injection of BCG plus LPS did not increase the mean survival time of mice that had been inoculated with tumor cells i.p. Sera from mice that had been treated with BCG plus LPS i.v. were cytotoxic for cultured tumor cells. These results seemed to indicate that growth-inhibitory effects of LPS on tumors inoculated s.c. were mediated by a humoral factor.

Suppressor T cells in BCG-treated mice interfere with an in vivo specific antitumoral immune response.

The interference by BCG in the induction and expression of a specific antitumoral immune reaction was studied in B6 mice, using the in vivo Winn assay and also active immunization. T cells immunized against MCA-induced fibrosarcoma (MC B6-1) transferred together with the tumour cells protected the syngeneic host against tumour take. Pretreatment of normal B6 mice with moderate or high doses of BCG prevented the development of a protective immune response after immunization. Moreover, a single dose of 1 mg, or 2 doses of 0.01 mg BCG, completely eliminated an established antitumour immunity. Suppressor cells are involved in the BCG-induced inhibitory effect; they interfered (1) with the expression of the antitumour response, since their addition to immune T cells in the Winn test resulted in decreased protection and (2) with the induction of the antitumour response, since injection of spleen cells from BCG-treated mice (BCG SpC) into normal mice before immunization inhibited the development of immunity. Treatment of BCG SpC with anti Thy 1.2 and anti Lyt 1.2 antibodies plus complement before injection into normal mice significantly decreased the suppressive activity, showing that the suppressor cells induced by BCG are T cells expressing the Lyt 1+ phenotype. The partial increase in protection obtained after IL-2 administration to BCG-treated mice suggests that the suppressive action of BCG SpC on the IL-2 producing capacity of helper T cells is only one of a number of possible mechanisms of T-cell-mediated suppression.

Lymphocyte Reactivity in Normal and Malignant Cell Proliferation Against a Phylogenetically Conserved Antigen in Fetal Extracts&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Lymphocyte reactivity to 3-M KCl extracts from fetuses of different species of origin was shown by the macrophage electrophoretic mobility (MEMB) and/or the leukocyte migration inhibition tests in tumor-bearing mice and humans. In chemical carcinogenesis, reactivity was detectable before tumors developed. Six weeks after sc injection of 1,000 micrograms benzo[a]pyrene [(BP) CAS: 50-32-8] into XVII/Bln mice, the MEMB test became positive. The latent period was 15 weeks after 1.0 micrograms BP, indicating a dose-response relationship of the phenomenon. Painting of mouse skin with 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6], croton oil (CAS: 8001-28-3), or benzene (CAS: 71-43-2) had the same sensitizing effect as BP. In contrast to the strong carcinogens BP and DMBA that caused lymphocyte reactivity to persist until tumors developed, benzene and the promoter croton oil induced only a transient effect. Termination of treatment abolished reactivity within 12 weeks. Lymphocyte reactivity to fetal extract in normal cell proliferation was evident from the fact that two-thirds-hepatectomized rats and BCG-treated mice became MEMB-positive. In hepatectomized rats the effect was reversible according to the completion of liver regeneration. Lymphocytes from tumor-bearing mice reacted with mouse and human fetal extract as well as with extracts from different developmental stages of frogs and fish. Fetal extracts were assumed to contain a phylogenetically conserved antigen.

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmete-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigenpresenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.

Mechanisms of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection.

The mechanism of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection was investigated by determining the effect of splenectomy, antithymocyte serum, and antimacrophage serum on resistance. It was greatly reduced by these treatments, not only in normal mice, but also in mice treated with live or heat-inactivated BCG. Production of circulating interferon by ectromelia virus and Newcastle disease virus was augmented in BCG-treated mice and was markedly depressed by splenectomy and antithymocyte and antimacrophage serum treatments in both BCG-treated and normal mice. Carbon clearance activity was activated in BCG-treated mice, but splenectomy did not influence phagocytic activity. These results suggest that augmented interferon production in the spleens of BCG-treated mice plays a major role in enhanced resistance. Other possible mechanisms are discussed.

Activation of peritoneal macrophages by concanavalin A or Mycobacterium bovis BCG for fungicidal activity against Blastomyces dermatitidis and effect of specific antibody and complement.

With a new short-term assay, where the reduction of CFUs in the inoculum could be measured, we investigated the killing of the dimorphic fungal pathogen Blastomyces dermatitidis in its yeast phase by murine peritoneal macrophages. Peritoneal macrophages from concanavalin A- or Mycobacterium bovis BCG-treated mice, but not resident or thioglycolate-elicited macrophages, significantly reduced the CFUs of B. dermatitidis in the inoculum. The activation of peritoneal macrophages for fungicidal activity by concanavalin A treatment was shown to be dose dependent and transient, i.e., absent after 72 h. These results indicate that it is possible for murine peritoneal macrophages to kill B. dermatitidis in vitro. The addition of specific antibody or complement or both did not enhance the killing of B. dermatitidis by these nonspecifically activated macrophages.

BCG-induced susceptibility of mice to challenge with Pseudomonas aeruginosa.

Mice infected with Mycobacterium bovis, BCG, were shown to be highly susceptible to subsequent challenge with Pseudomonas aeruginosa. The susceptibility was characterized by the enhanced mortality and shortened survival after challenge with P. aeruginosa. BCG-treated mice did not show any enhanced susceptibility to challenge with Gram-positive bacteria such as Staphylococcus aureus or Listeria monocytogenes. BCG-treated mice eliminated P. aeruginosa from their organs in a pattern similar to that in untreated mice. There was no significant difference in the bactericidal activities of polymorphonuclear cells and macrophages between BCG-treated and untreated mice. An equal amount of endotoxin was detected by the Limulus lysate assay in the blood of both BCG-treated and untreated mice after challenge with P. aeruginosa. The enhanced susceptibility induced by BCG pretreatment could be decreased when the mice were rendered LPS-tolerant by injections of small amounts of LPS. These results suggest that BCG-induced susceptibility to P. aeruginosa can be ascribed to an enhanced susceptibility to the lethal effect of LPS produced by challenge bacteria, and not to the impairment of the ability to eliminate infected bacteria.

Possible role of macrophage-like suppressor cells in the anti-tumour activity of BCG.

The i.v. injection of high doses (3 mg) of BCG into C3H mice bearing a transplantable 3-methylcholanthrene-induced fibrosarcoma caused the regression of a significant proportion. This effect was most evident when the BCG was injected on the day of the graft, or 7 days later. The injection of this agent either 14 days before the graft, or in low doses (0.1 or 0.5 mg), or directly into the tumour (i.t.) only prolonged the survival of the animals. Spleen cells from systemic high-dose BCG-treated mice were found to exert a strong nonspecific cytostatic effect in vitro that was not an artefact of the test conditions, and was not expressed by cells from low-dose animals. The cytostatic effect was shown to be caused by cells with the characteristics of macrophages, i.e. they were strongly adherent, unaffected by treatment with anti-Thy 1.2 + C', radioresistant but heat-sensitive, and were detected in BCG-treated "B" mice. The spleens of high-dose BCG-treated mice also contained suppressor cells that were capable of inhibiting the in vitro reactivity of normal T cells to PHA. Like the cytostatic effect, this suppressor activity was not detected in low-dose mice, and the cells responsible had the properties of macrophages; the effect was lost after the removal of adherent cells by sequential exposure to plastic and colloidal iron, but was conserved after treatment with anti-Thy 1.2 + C'. T-cell-deprived animals, such as "B" or nude mice, also developed suppressor-cell activity when treated with systemic high-dose BCG. Close parallels became evident between the in vivo anti-tumour activity of BCG, the in vitro cytostatic effect, and the suppressor-cell activity. We here discuss the possible role of suppressor cells in the mechanism of action of this agent.

Corynebacterium parvum treatment of P388 tumor-bearing mice. II. Lysosomal enzyme levels associated with P388 tumor cells.

The number of tumor cells recovered form the peritoneal cavity of mice administered Corynebacterium parvum (C. parvum), Bacillus Calmette-Guérin (BCG) or saline 2 days before tumor implant was assessed on days 1, 2, 3, 5 and 7 following the intraperitoneal (ip) administration of 10(6) P388 leukemic cells. C. parvum-treated mice manifested a significant decrease in the number of tumor cells recovered from the peritoneal cavity on days 3-7, while BCG-treated mice had tumor cell yields comparable to saline control values. Lysosomal enzyme activity (acid phosphatase and beta-glucuronidase) in the nonadherent lavage cell population, which comprised tumor cells and host cells obtained form tumor-bearing animals, closely reflected the changes observed in tumor cell numbers and was initially assumed to be associated with tumor cells. When lysosomal enzyme activity was expressed as a function of the number of tumor cells or cellular protein, an enhanced activity following C. parvum treatment but not following BCG treatment was demonstrated. Enzyme activity associated with tumor cells was maximal 2 days prior to the profound depression in tumor cell maximal 2 days prior to the profound depression in tumor cell yield and may be associated with tumor cell killing. It is concluded that a correlation may exist between lysosomal enzyme activity, tumor cell numbers and the protective effect of the immunostimulant C. parvum. Whether the correlation is direct or indirect remains to be resolved.

Altered biodistribution of radiogallium following BCG treatment in mice

Abstract A mouse animal model has been used in this study which was prompted by clinical observations that 67 Ga-citrate scintigraphs of patients with multi-system melanoma who had been treated with BCG revealed unusually high blood concentrations of the radiotracer, abnormal diffuse pulmonary localization and increased uptake at sites not corresponding with clinical evidence of metastases. Groups of mice were immunized by a series of intradermal injection of BCG. Tissue distribution studies were performed 48 h after i.v. injection of 67 Ga-citrate. These same tissues were also examined histopathologically. Increased uptake of radiogallium was seen in the liver, lungs, spleen, kidneys and blood of the BCG-treated mice when compared to normal controls. A close correlation was seen between the incidence of increased radiogallium concentrations and the presence of microscopically visible lesions in each of the tissue samples. Autoradiography of liver sections from BCG-treated mice injected with 67 Ga-citrate indicated that the radioactivity was localized to a large extent, at sites of BCG induced granuloma. 67 Ga was shown to be taken up significantly by live BCG cells in in vitro incubation but little or no incorporation occurred with heat-killed BCG.

BCG-induced macrophage suppression in mice: Suppression of specific and nonspecific antibody-medicated and cellular immunologic responses

The intravenous injection of killed BCG in an oil-in-saline emulsion (BCG-E) results in the development of intense chronic granulomatous inflammation in the lungs and spleen of C57B1/6 (B6) but not CBA mice. B6 mice injected intravenously with BCG-E exhibited marked suppression of antibody responsiveness and delayed hypersensitivity to sheep erythrocytes, as well as proliferation in response to PPD. In contrast, CBA mice similarly treated with BCG-E were not suppressed in any of these reactivities. The spleen is an important organ in this phenomenon since suppression was reversed by splenectomy and could be transferred to normal recipients with spleen cells from BCG-treated mice. Spleen cells responsible for suppression were adherent to plastic petri plates, removed with carbonyl iron, and were not eliminated with either anti-Thy-1 or anti-immunoglobulin serum + C. This study indicates that macrophages from BCG-inflamed spleen are capable of potent suppression of both antibody- and cellular-mediated immunologic reactivity.