Enhanced elimination of Listeria monocytogenes at the site of delayed footpad reaction.

The protective activity against a challenge infection with Listeria monocytogenes was investigated at the site of a delayed footpad reaction in mice immunized with viable or killed listeria. Delayed footpad reactivity was induced only in mice immunized with viable bacteria. Rapid and marked elimination of challenge bacteria was observed only at the site of reaction in mice immunized with viable bacteria but not in mice immunized with killed bacteria. Macrophage migration inhibitory activity was observed equally in both groups of mice. These results suggest that the delayed footpad reaction contributes directly to the elimination of bacteria irrespective of macrophage migration inhibitory activity.     

Relationship between non-specific activity of macrophages and immune responses to Listeria monocytogenes.

Delayed footpad reactions and acquired cellular resistance to Listeria monocytogenes were studied in mice whose mononuclear phagocyte system (MPS) had been blocked or stimulated. Colloidal carbon was used for the blockade of MPS and Corynebacterium parvum used for the stimulation. Strong delayed footpad reactions. On the other hand, the i.v. injection of 3 X 10(1) listeria induced an appreciable level mice, while in MPS-stimulated mice, i.v. injection with even 4 X 10(3) listeria could not induce such strong delayed footpad reaction. On the other hand, the i.v. injection of 3 X 10(1) listeria induced an appreciable level of delayed footpad reaction only in MPS-blocked mice. Acquired cellular resistance was depressed by MPS stimulation, whereas it was augmented by MPS blockade. These results suggested that non-specific activity of MPS modulates subsequent immune responses after inoculation of listeria.     

Local reactivity, local resistance and systemic dissemination in Mycobacterium lepraemurium (MLM) infection.

Local reactivity measured as swelling of the infected footpad, local resistance to bacterial multiplication, and capacity to limit systemic dissemination were studied in C57BL, C3H/Bom, C3H/HeJ, and A/Sn mice inoculated with Mycobacterium lepraemurium. C57BL mice developed a strong local reaction with a sudden onset, and effectively limited local multiplication as well as systemic dissemination of bacteria to the liver and spleen as determined 19 weeks after the inoculation. C3H/Bom mice showed no local reaction, had high numbers of bacteria locally, and had extensive systemic dissemination of the infection. C3H/HeJ mice, on the other hand, developed a small local reaction and had less systemic dissemination of bacteria than C3H/Bom mice. In C57BL mice and in the two C3H substrains local reactivity, local resistance to infection, and resistance to systemic spread of the infection paralleled each other. In contrast, A/Sn mice showed a small local reaction, had the most extensive bacterial multiplication at the site of inoculation of the four mouse strains tested, and at the same time were the mice that most effectively restricted systemic dissemination of the infection. Thus, the mechanisms restricting local bacterial multiplication may be different from the mechanisms limiting bacterial dissemination. Neither bacterial growth locally at the site of subcutaneous inoculation in the footpad, nor systemic dissemination of the infection, followed a mouse strain pattern consistent with the Ity/Lsh/Bcg gene model. In experimental mycobacterial infection both local bacterial growth at the site of inoculation and systemic dissemination should be determined.     

Expression of antibacterial resistance at the site of a delayed hypersensitivity reaction.

The site of a delayed hypersensitivity reaction to tuberculin or bovine serum ablumin was shown to contain mechanisms that expressed increased antibacterial activity, as evidenced by restricted growth of a local inoculum of Listeria monocytogenes. As was the case with a delayed hypersensitivity reaction, the local generation of antibacterial activity was antigen specific and T-cell dependent. Antibacterial resistance was always expressed at the site of injection of specific antigen in sensitized mice, even though under certain circumstances there was no measurable increase in footpad thickness at this site. It thus appears that nonspecific antibacterial resistance represents a sensitive and quantitative method for measuring delayed hypersensitivity. More importantly, this study serves to provide a functional meaning for the delayed-type hypersensitivity reaction in that it demonstrates that such a reaction causes the focusing of mechanisms that can restrict the growth of bacteria at a site which may represent a source of microbial invasion.     

Transfer of resistance to primary infection of Listeria monocytogenes and early induction of delayed hypersensitivity by sera from L. monocytogenes-infected mice.

We found a new phenomenon which differs from previous reports on experimental listeriosis, that is, failure of passive transfer of serum from Listeria monocytogenes-infected mice to convey resistance to the bacterium. Transfer of immune serum from L. monocytogenes-infected mice markedly augmented resistance to the bacterium, and mechanisms of the transfer of L. monocytogenes-immune serum were investigated. Transfer of immune serum prevented L. monocytogenes lethality. This effect of the immune serum was transferred dose dependently. Augmentation of resistance to L. monocytogenes also appeared in elimination of bacteria from the spleen. The growth of bacteria within 2 days in the spleen was not inhibited. Transfer of the immune serum augmented and accelerated induction of a delayed footpad reaction. Delayed hypersensitivity-dependent accumulation of mononuclear cells, detected by focus formation reaction in the liver, was also augmented. In contrast, polymorphonuclear cell accumulation in the liver was suppressed. Development of delayed hypersensitivity reactions was correlated with the elimination of bacteria in the spleens. These effects of the immune serum were expressed antigen specifically; however, the effector molecule(s) in the immune serum differs from immunoglobulin molecules.     

Local immune response to Mycobacterium lepraemurium in C3H and C57Bl/6 mice.

Subcutaneous footpad inoculation of living M. lepraemurium (L.MLM) induced, in high responder C57Bl/6 mice, a local granulomatous reaction associated with the production of effector cells which stopped the multiplication of bacilli in the draining popliteal node with the concurrent development of 24--48 hr delayed type hypersensitivity (DTH). The thymus-dependent local reaction did not occur after the injection of heat-killed M. lepraemurium (HK.MLM) or after the inoculation of L.MLM in nude mice. However, HK.MLM injection interfered with the onset of the local reaction and enhanced acid-fast bacteria (AFB) counts in the draining node. In low responder C3H mice, L.MLM produced a local and delayed footpad swelling but no restriction of bacilli multiplication in the draining lymph node was observed. This unresponsiveness was not due to an overloading of the inoculum dose since doses ranging from 3 x 10(4) to 3 x 10(7) MLM did not produce any granulomatous local reaction as in C57Bl/6 mice. The injection of dead bacilli in the contralateral footpad of subcutaneously (s.c.) infected C3H mice revealed Arthus-like and 18--24 hr delayed reactions. When 10(6) L.MLM per mouse were injected intravenously (i.v.), systemic infection, measured in the spleen, was found to be less restricted in C57Bl/6 than in C3H mice. Moreover, in C57Bl/6 mice low doses of L.MLM injected i.v. delayed the local reaction at first, then enhanced footpad swelling and AFB counts in the draining nodes, indicating some acquired defect of peripheral immunity. When a high dose of L.MLM (2 x 10(8)/mouse) was injected i.v., C57Bl/6 mice died sooner than C3H mice, indicating certain discrepancies between local resistance and systemic susceptibility.     

Role of specific delayed-type hypersensitivity in Pseudomonas aeruginosa-infected mice

The role of specific cell-mediated immunity was studied in mice injected in the hind footpad with viable Pseudomonas aeruginosa cells. The results reported here show that a state of specific delayed-type hypersensitivity, evaluated both as footpad swelling and as weight increase of popliteal lymph node, occurs in P. aeruginosa-infected mice. Furthermore, a T-cell-enriched spleen population from infected animals was able to transfer delayed hypersensitivity to normal recipients. However, identity at the major histocompatibility complex to transfer delayed hypersensitivity was required. Acquired cellular resistance was not transferred to normal recipients by immune T lymphocytes. On the contrary, mice receiving immune T cells showed an increase in the severity of the lesion caused by a viable challenge. The dichotomy between acquired cellular resistance and delayed hypersensitivity, and the possibility that T-cell reactivity to P. aeruginosa may be actively controlled, is discussed.     

Delayed hypersensitivity to Staphylococcus aureus in mice: in vivo responses to isolated Staphylococcal antigens.

The development of delayed hypersensitivity (DH) to Staphylococcus aureus in Swiss mice was evaluated by the footpad (FP) assay. In order to determine which component of the bacteria was responsible for the in vivo immune reactivity, purified Staphylococcal cell wall, cell membrane, protein A, lipoteichoic acid, teichoic acid, as well as lipid-free membrane proteins were isolated. The immune responses of mice receiving one to eight S. aureus injections indicated that the first DH peak, following three injections, was primarily dependent upon protein antigens associated with the bacterial membrane. Increased bacterial injections gave rise to a second DH peak following seven injections which was dependent upon multiple bacterial components including cell wall, protein A, and membrane proteins.     

The in vivo effects of concanavalin A on the cellular immune response in BALB-c mice. Inhibition of the delayed hypersensitivity reaction to tuberculin

The suppressive effect of concanavalin A (Con A) on the cell-mediated immune response was demonstrated in BALB/c mice made immune to challenge with tuberculin. 400 μg of Con A administered intraperitoneally simultaneously with injection of antigen into the footpad of primed animals resulted in suppression of the delayed hypersensitivity reaction as detected by a sensitive radioisotopic footpad assay. If Con A was given in a dose of 100 μg three times weekly to normal mice being sensitized to tuberculoprotein, the maximum immune response following complete immunization was significantly suppressed but not abolished. The suppressive effect of Con A appeared to be due to its binding to cell membranes, since splenic lymphocytes obtained from Con A-treated immune mice failed to respond in the local adoptive transfer reaction to tuberculin. The lymphocytes regained their immune reactivity to tuberculin if they were first incubated in α-methyl-D -mannoside. These findings represent the first demonstration of in vivo immunosuppression by Con A of the tuberculin immune response in the mouse. Both the inductive and established responses of the delayed hypersensitivity reaction appear to be affected. The reversible inhibition of the local adoptive transfer of tuberculin immunity suggests the depression of the response is due to binding to or alteration of antigen receptor sites by Con A.     

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.     

Correlation of Increased Metabolic Activity, Resistance to Infection, Enhanced Phagocytosis, and Inhibition of Bacterial Growth by Macrophages from Listeria- and BCG-Infected Mice

Macrophages from mice infected with facultative intracellular organisms such as Listeria monocytogenes and BCG have been shown to resist infection by antigenically unrelated intracellular bacterial parasites. This study compares phagocytosis, bacterial growth inhibition, and oxidation of glucose by macrophages from normal mice, mice infected with listeria or BCG, or mice immunized with killed listeria in incomplete Freund's adjuvant. Macrophages from listeria- and BCG-infected mice ingested more listeria; 67 and 57%, respectively, had three or more cell-associated bacteria versus 22% of controls (P < 0.001). Peritoneal macrophages from listeria- and BCG-infected animals significantly (P < 0.001 covariance analysis) inhibited growth of listeria in suspension, whereas control macrophages had no such inhibitory effect. The rate of oxidation of glucose-1-(14)C was higher in macrophages from listeria- and BCG-infected mice than from either uninfected animals or those immunized with killed listeria. During phagocytosis of killed or live bacteria, or latex particles, the rate of glucose oxidation was increased (P < 0.01). These data suggest that the cellular immunity after infection by an intracellular organism is associated with an increase in metabolic activity of macrophages, namely, an increase in the rate of glucose oxidation resulting in enhancement of phagocytosis and killing.     

The induction of renal autoantigen-specific T cells by a local Listeria monocytogenes infection.

In order to examine the possibility that a local chronic infection can induce organ-specific autoimmune disease, we inoculated unilateral kidneys with viable Listeria monocytogenes (intrarenal infection). The delayed footpad reaction against syngeneic kidney homogenate (KH) became positive from 1 week after initiating the intrarenal infection. A proliferative response of the spleen T cells from the infected mice was also observed against KH from 1 week after initiating the intrarenal infection, but no such response was seen against liver homogenate (LH). In contrast, an intravenous Listeria infection did not induce a delayed footpad reaction or proliferative response against KH, suggesting that these autoimmune responses were not caused by molecular mimicry between renal antigens and Listeria antigens. Furthermore, the ability to transfer the autoimmune response of spleen T cells from intrarenally infected mice was examined. The transferred mice showed a positive delayed footpad reaction against KH and an interstitial infiltration of mononuclear cells in their kidneys. These results demonstrate that the intrarenal Listeria infection induced renal autoantigen-specific T cells, which subsequently induced an autoimmune interstitial nephritis (AIN). The autoreactive T cells were all induced without immunization by autoantigens mixed with complete Freund's adjuvant. Based on these findings, we propose that a local bacterial infection may induce an autoimmune response against autoantigens in the infected organ and subsequently trigger organ-specific autoimmune disease.     

Delayed hypersensitivity to Trypanosoma cruzi in mice: specific suppressor cells in chronic infection.

In mice chronically infected with Trypanosoma cruzi there are antigen-specific suppressor cells which inhibit the development of delayed hypersensitivity (DTH) to T. cruzi antigen but not to an unrelated antigen, keyhole limpet haemocyanin (KLH). This was shown by comparing the 24 hr footpad reactivity, elicited by injection of soluble T. cruzi antigen, of infected mice with that of mice immunized with killed T. cruzi antigen after pretreatment with cyclophosphamide. In the latter, 24 hr footpad swelling represented a DTH reaction in that the cellular infiltrate was predominantly mononuclear and the reactivity could be transferred to normal recipients by lymphoid cells but not by serum. Chronically-infected mice also developed 24 hr footpad swelling but the fact that this was undiminished from an earlier 3 hr reaction and could not be transferred to normal recipients by either local or systemic injection of cells, as well as the histological features, all implied that it represented a prolonged Arthus reaction. The absence or minimal levels of specific DTH detectable in chronic T. cruzi infected mice was accompanied by the presence in their spleens of cells which specifically suppressed the generation of DTH to T. cruzi in normal mice. Suppressor cell activity was radioresistant (10 Gy/1000 rad) and T-cell mediated as defined by significantly decreased and increased suppression following anti-Thy 1.2 serum treatment and nylon wool fractionation, respectively. The ability of chronic T. cruzi mice to develop DTH to an unrelated antigen KLH was unimpaired.     

A lack of correlation between antigen-specific cellular reactions and resistance to Mycobacterium lepraemurium infection in mice.

Following infection subcutaneously in the footpad with 10(7) Mycobacterium lepraemurium organisms C57BL mice were able to limit multiplication of organisms at the infection site for the 6 months studied and to limit organism spread to the draining lymph node. Large numbers of organisms were present in the footpad and draining lymph node of BALB/c mice at 6 months. In spite of this difference in local immunity the changes in cellular reactivity to specific antigen as assessed by the delayed footpad response and the in vitro proliferative response of draining lymph node cells were similar in the two strains over the time studied.     

Protective Effect of a Traditional Chinese Medicine, Xiao-Chai-Hu-Tang (Japanese Name: Shosaiko-To), On Listeria Monocytogenes Infection in Mice

Lethal effect of Listeria monocytogenes (L. monocytogenes) in mice was prevented by an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), 4 days before ip bacterial infection. The numbers of bacteria in the peritoneal cavity and liver were smaller in shosaiko-to-treated mice from one day after the infection. Macrophage accumulation in the peritoneal cavity after ip inoculation of L. monocytogenes was observed in both untreated and shosaiko-to-treated mice. Although rates of such increases were almost the same between both groups, the absolute number of macrophages was larger in shosaiko-to-treated than in untreated mice because of a higher level of the macrophage number at 4 days after ip injection of shosaiko-to. In untreated mice, bactericidal activity of peritoneal macrophages decreased from one day to 3 days after ip injection of killed L. monocytogenes. Such an activity was maintained at the same level from 1 to 3 days in shosaiko-to-treated mice. Augmented accumulation of macrophages and maintenance of their bactericidal activity may be main mechanisms of the augmented resistance in shosaiko-to-treated mice. Augmented resistance against bacterial growth in the thigh muscle in ip shosaiko-to-treated mice may be caused by such mechanisms. The effect of shosaiko-to observed at an early stage of infection may be T cell-independent, since such an effect was observed in athymic nude mice and delayed footpad reaction could not be detected at such a timing in euthymic normal mice.     

Relationships among differentiated T-cell subpopulations. IV. Preferential induction of delayed footpad reaction to xenografts and its radioresistant nature.

Immune response in mice to xenogeneic cells was characterized by positive delayed footpad reaction and negative macrophage migration inhibition. Mice immunized with allogeneic cells exhibited negative delayed footpad reaction and positive macrophage migration inhibition. Cytotoxic T lymphocytes were detected only in mice immunized with allogeneic tumour cells. Delayed footpad reaction against xenogeneic cells was radioresistant and the immune lymphocytes responsible for such a reaction were presumed to have some relation to xenograft rejection.     

Generation of Listeria monocytogenes-specific T cells mediating delayed footpad reaction and protection in neonatally thymectomized mice but not in nude mice

Neonatally thymectomized (NTx) mice, sham-operated control mice and congenitally athymic nude mice were immunized with viable Listeria monocytogenes and their spleen cells examined for the capacity to transfer both delayed footbad reaction and protection against challenge at the site of local transfer. Cells from immune NTx mice conferred significant degrees of delayed footpad reaction and protection comparable to sham mice, while cells from immune nude (nu/nu) mice did not. This abilty was completely eliminated by the treatment of cells with anti-Thy1, anti-Lytl or anti-L3T4 antibody plus complement but not with anti-Lyt2 antibody plus complement. These results indicated that NTx mice can normally mount the immunity to L. monocytogenes by generating Lyt1⁺2^–, L3T4⁺ T cells. Immune competence of NTx mice and thymus dependency of various immune responses are discussed.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction

We previously found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the culture supernatant of the mixture of immune spleen cells and erythrocyte antigen, or in the serum of mice immunized with heterologous erythrocytes and exhibiting delayed-type footpad reaction. To elucidate whether this kind of factor (DTH-augmentation factor; DAF) participates in the establishment of DTH to various kinds of antigen besides erythrocyte antigen, we chose a bacterial antigen, Listeria monocytogenes, which is a facultative intracellular bacterium. In the present study, we demonstrated that the immune serum from mice immunized with viable Listeria augmented the delayed-type footpad reaction to Listeria. Furthermore, acquired resistance against Listeria was also augmented by the transfer of such immune serum. Such augmentation of acquired resistance was observed in sites infected locally and in the spleen of mice infected systemically. This effect was also seen in sera from mice immunized with heat-killed Listeria emulsified with complete Freund's adjuvant.     

Protective Effect of a Traditional Chinese Medicine,Xiao-Chai-Hu-Tang (Japanese Name: Shosaiko-To), On Listeria Monocytogenes Infection in Mice

Abstract

Lethal effect of Listeria monocytogenes (L. monocytogenes) in mice was prevented by an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), 4 days before ip bacterial infection. The numbers of bacteria in the peritoneal cavity and liver were smaller in shosaiko-to-treated mice from one day after the infection. Macrophage accumulation in the peritoneal cavity after ip inoculation of L. monocytogenes was observed in both untreated and shosaiko-to-treated mice. Although rates of such increases were almost the same between both groups, the absolute number of macrophages was larger in shosaiko-to-treated than in untreated mice because of a higher level of the macrophage number at 4 days after ip injection of shosaiko-to. In untreated mice, bactericidal activity of peritoneal macrophages decreased from one day to 3 days after ip injection of killed L. monocytogenes. Such an activity was maintained at the same level from 1 to 3 days in shosaiko-to-treated mice. Augmented accumulation of macrophages and maintenance of their bactericidal activity may be main mechanisms of the augmented resistance in shosaiko-to-treated mice. Augmented resistance against bacterial growth in the thigh muscle in ip shosaiko-to-treated mice may be caused by such mechanisms. The effect of shosaiko-to observed at an early stage of infection may be T cell-independent, since such an effect was observed in athymic nude mice and delayed footpad reaction could not be detected at such a timing in euthymic normal mice.     

Induction and specificity of delayed hypersensitivity to Staphylococcus aureus in mice.

The induction and specificity of delayed hypersensitivity (DH) to Staphylococcus aureus in mice was evaluated in vivo by the footpad (FP) assay and in vitro by spleen cell stimulation. Repeated infections result in a biphasic DH response. The first DH response, observed following three subcutaneous injections, was route and antigen specific, required viable organisms, and could not be enhanced by the incorporation of bacteria in adjuvants. Footpad reactivity was transferred to non-injected recipients by spleen cells but not serum and was inhibited by anti-thymocyte serum but not by cyclophosphamide. Spleen cell stimulation was maximal with homologous antigen, but, some cross reactivity was observed when cells were stimulated with hererologous gram-positive antigens. No cross reactivity was observed when antigens from gram-negative bacteria were used to stimulate spleen cells. The FP reactivity to homologous antigen following 7 injections, the second DH response, is of longer duration than that following 3 injections. Mice given seven injections exhibit a greater degree of cross reactivity to heterologous gram-positive but not gram-negative bacterial antigens. Similar results were observed when spleen cells from mice receiving 7 injections were simulated with gram-positive antigens. Furthermore, the degree of spleen cell stimulation following three of seven injections could be increased by elicitation prior to the vitro experiments.