Footpad Reaction Research Articles

Differences in thymus dependency among the alloreactive T-cell subpopulations in their development: Graft-versus-host reaction appears to be mediated by delayed-type hypersensitivity of tuberculin type

The effects of thymectomy at various times after birth, 1 and 7 days (Tx-1, Tx-7), on delayed footpad reactions, cell-mediated cytotoxicity (CMC), production of migration inhibitory factor (MIF), and graft-versus-host reactions (GVHR) against allogeneic antigens were determined by experiments with 8-week-old mice. Delayed footpad reactions were detected in Tx-1 mice, but CMC, production of MIF, and the ability to raise GVHR were not detected in such mice. CMC was detected in Tx-7 mice. However, the ability to raise GVHR and to produce MIF was abolished by thymectomy at 7 days after birth. These results suggest that the effector mechanism to mount a GVHR might be mediated by delayed-type hypersensitivity of tuberculin type rather than CMC to host antigens.

Immunomodulating activity of thymosin fraction 5 and thymosin alpha 1 in immunosuppressed mice.

We found that both thymosin from calf thymus and its constituent peptide alpha 1 prepared by chemical synthesis restore cell-mediated immunity following its suppression in mice by injection of 5-FU. Conditions suitable for assessing the thymosin activity by means of footpad reaction were established in such immunosuppressed mice. In this new animal model, thymosin alpha 1-peptide showed activity at a low dose of 5-50 micrograms/kg, which was 100-1,000 times less than that required for thymosin F-5 preparations. Further studies utilizing the adoptive transfer technique showed that alpha 1-peptide corrects the 5-FU-induced suppression of mature T cells, transferring the DTH response as well as that of macrophage function responsible for the expression of footpad reaction. Furthermore, regeneration of lymph node and bone marrow cells as well as CFU-c (progenitor cells of macrophages and granulocytes) was enhanced by thymosin alpha 1 in the 5-FU-treated mice. All these results indicate that thymosin alpha 1 accelerates the replenishment and maturation of haematopoietic cells, including not only T cells but also macrophages, when they have been severely damaged by the 5-FU treatment.

H-2-linked gene(s) influence the granulomatous reaction to viable Mycobacterium lepraemurium in the mouse.

The genetic control of the granulomatous response to viable Mycobacterium lepraemurium (MLM) was studied in C3H and C57BL/6 inbred strains, BXH recombinant inbred strains, (C3H X C57BL) F1 hybrids, and backcross mice. The results indicate that an autosomal dominant gene, or linked complex of genes, has a marked influence on the footpad reaction to viable MLM. The distribution of responders and non-responders among 12 BXH recombinant inbred strains and linkage analysis in C3H X (C3H X C57BL)F1 backcross mice indicated that the response gene(s) are linked to the H-2 complex on chromosome 17. The same gene(s) also influence host restriction of MLM multiplication and thus appear to be the first H-2-linked gene(s) influencing resistance to a bacterial infection.

Restoration by levamisole of immune responses suppressed in tumor-bearing mice.

Restorative effects of levamisole on suppressed immune responses were observed in BALB/c mice inoculated with a syngeneic methylcholanthrene-induced fibrosarcoma and immunized with nucleated chicken erythrocytes. In tumor-bearing mice, cell-mediated cytotoxicity, delayed footpad reaction, and antibodies were suppressed, and only the delayed footpad reaction was restored by levamisole. Suppressive effects in tumor-bearing mice can be transferred with sera or spleen cells, and the expression of suppressive effects cannot be abolished by treatment of the recipients with levamisole. However, the generation of suppressor mechanisms was affected by treatment of tumor-bearing donors with levamisole.

Macrophage procoagulant activity as a measure of cell-mediated immunity in the mouse.

Thioglycollate-induced peritoneal exudate cells (TG-PEC) developed increased procoagulant activity after incubation with lymphokine and lipopolysaccharide (LPS). Dilutions of up to 1/1000 for insoluble Con A and 1/200 for periodate-induced lymphokine supernatants were active in enhancing macrophage procoagulant activity (MPCA), which was detected after a 2-hr incubation period and steadily increased over 20 hr. MPCA could also be induced by antigen; peritoneal cells from sensitized B6AF1 mice with strong footpad reactions to ovalbumin (OVA) responded to as little as 0.1 microgram/ml OVA in the MPCA test in an antigen-specific manner. By contrast, PEC from sensitized CBA/J mice that gave poor in vivo responses to OVA only reacted with high concentrations of the antigen in vitro. Production of the lymphokine responsible for induction of MPCA required an Ly-1+2- T cell, a nylon wool-adherent cell, and an la-17-bearing adherent cell. The MPCA induced by lymphokine or LPS did not appear to be a serine esterase and was not inhibited by phospholipase C. Coagulation of human factor-deficient plasma with activated TG-PEC indicated a requirement for Factor X.

Properties of cloned T cells that mediate delayed-type hypersensitivity against ovalbumin in mice

Cloned T-cell lines that mediate delayed-type hypersensitivity (DTH) against soluble protein antigen, ovalbumin (OA), were established in (C57BL/6 X DBA/2)F 1 mice and their properties were examined. They induced antigen-specific delayed-type footpad reactions, characterized histologically by a predominant mononuclear cell infiltration, when transferred intravenously into syngeneic mice. Morphologically, they were medium or large lymphoblasts with granules in the cytoplasm and expressed Lyt 1 cell surface antigens. One of them proliferated antigen specifically under the presence of both C57BL/6 and F 1 accessory cells, while others proliferated antigen specifically only under the presence of F 1 accessory cells. They also produced macrophageactivating factor (MAF) and substances which mediate a DTH-like footpad inflammatory reaction with a maximum 6 hr after injection into the footpad of normal mice, when incubated in the presence of specific antigen and specific accessory cells in a serum-free medium for 24 hr. These results demonstrate that cloned DTH-effector T cells, established here against soluble protein antigen, are Lyt 1-positive, large lymphoblasts and that they produce MAF and footpadreactive inflammatory substances antigen specifically under the presence of specific accessory cells.

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium.

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.

IMMUNOPHARMACOLOGICAL STUDIES OF SODIUM COPPER CHLORPHYLLIN (SCC)

Effect of sodium copper chlorophyllin (SCC) on experimental allergic reaction was investigated. IgE antibody mediated reactions, homologous passive cutaneous anaphylaxis (PCA) in rats and the release of anaphylactic mediators (histamine and/or slow reacting substance of anaphylaxis (SRS-A)) from sensitized guinea pig lung tissues or rat peritoneal mast cells classified as a Type I reaction were clearly inhibited by SCC at a similar potency as N-(3’,4’-dimethoxy cinnamoyl) anthranilic acid (N-5’). The increase of vascular permeability in rat skin caused by autacoids or enzymes that participate in the Type I reaction was also inhibited by SCC. Type II or III, complement dependent, reactions including reversed cutaneous anaphylaxis (RCA) in rats and Forssman cutaneous vasculitis (FCV) in guinea pigs were inhibited by SCC. Prednisolone inhibited RCA in rats, but did not inhibit FCV in guinea pigs. Two experimental types of glomerulonephritis, nephrotoxic serum (NTS) nephritis in rats and immune complex nephritis in (NZW×NZB) F1, mice, in which Type II and III reactions might participate in the onset and the development of the disease, were slightly inhibited by SCC in terms of the biochemical changes of blood and urine parameters and histopathological scores. A moderate remission of the onset and development of these two experimental types of nephritis was recognized by the administration of prednisolone. Delayed hypersensitivity reaction as a Type IV reaction caused by sheep red blood cells (SRBC) in sensitized mouse footpad was not affected by SCC. Prednisolone clearly inhibited the SRBC induced footpad reaction in mice. IgM antibody production in mice and IgE antibody production in rats were not influenced by daily injection of SCC.

ベリリウム化合物による細胞性免疫についての検討

In order to examine the sensitizing ability of beryllium (Be), a footpad reaction test and a macrophage migration inhibition test were carried out using mice treated with beryllium compounds. The findings of the experiments are summarized as follows: 1) The footpad reaction test of the mice injected with BeCl2 once a week subcutaneously at their dorsal area showed the positive results after four injections. The macrophage migration inhibition test was also performed by an agarose plate method using peritoneal cells and spleen cells obtained from these mice. The ratio of the number of positive mice showed more than 50% after five injections when peritoneal cells were used and after six injections when spleen cells were used. 2) The footpad reaction test of mice injected with one of various Be-compounds six times once a week showed a cross positive reaction against these compounds, and the macrophage migration of these mice was inhibited by these Be-compounds, but not by other metals. 3) When mice were once injected with 7BeCl2, about a half dose of the injected metal was discharged from the body within 7 days after the injection, and about a half dose of the residual metal was still found in the injected site. Further investigations may be required to solve the point that the persistent remaining of this metal in situ may raise the Be sensitizing ability.

Immunopharmacological studies of sodium copper chlorphyllin (SCC).

Effect of sodium copper chlorophyllin (SCC) on experimental allergic reaction was investigated. IgE antibody mediated reactions, homologous passive cutaneous anaphylaxis (PCA) in rats and the release of anaphylactic mediators (histamine and/or slow reacting substance of anaphylaxis (SRS-A] from sensitized guinea pig lung tissues or rat peritoneal mast cells classified as a Type I reaction were clearly inhibited by SCC at a similar potency as N-(3',4'-dimethoxy cinnamoyl) anthranilic acid (N-5'). The increase of vascular permeability in rat skin caused by autacoids or enzymes that participate in the Type I reaction was also inhibited by SCC. Type II or III, complement dependent, reactions including reversed cutaneous anaphylaxis (RCA) in rats and Forssman cutaneous vasculitis (FCV) in guinea pigs were inhibited by SCC. Prednisolone inhibited RCA in rats, but did not inhibit FCV in guinea pigs. Two experimental types of glomerulonephritis, nephrotoxic serum (NTS) nephritis in rats and immune complex nephritis in (NZW X NZB) F1 mice, in which Type II and III reactions might participate in the onset and the development of the disease, were slightly inhibited by SCC in terms of the biochemical changes of blood and urine parameters and histopathological scores. A moderate remission of the onset and development of these two experimental types of nephritis was recognized by the administration of prednisolone. Delayed hypersensitivity reaction as a Type IV reaction caused by sheep red blood cells (SRBC) in sensitized mouse footpad was not affected by SCC. Prednisolone clearly inhibited the SRBC induced footpad reaction in mice. IgM antibody production in mice and IgE antibody production in rats were not influenced by daily injection of SCC.

Resistance and susceptibility to infection in inbred murine strains: I. Variations in the response to thymic hormones in mice infected with Candida albicans

Nine inbred murine strains were either highly resistant or highly susceptible to intravenous challenge with 4 × 10 4 to 1 × 10 5 cells of Candida albicans. The resistant strains had the capacity to develop delayed footpad reactions on appropriate sensitization and challenge; the susceptible strains did not have this innate capacity. Administration of thymosin fraction 5 beginning on the day of infection greatly increased the resistance of the susceptible strains to infection, but decreased the resistance of the resistant strains. In contrast, thymosin fraction 5 enhanced the delayed footpad responses of resistant-sensitized mice to specific antigen, but did not have a detectable effect on the delayed footpad reactions of the susceptible strains. Reinfection of the two types of strains had different effects, in that, depending on the strain, resistance could be increased, decreased, or not influenced at all.

Development of Immunity against Listeria monocytogenes in athymic Nude versus neonatally thymectomized mice

The thymus requirement for the development of immunological responsiveness was determined by estimation of immune responses raised to Listeria monocytogenes in athymic nude, neonatally thymectomized, and sham-operated mice at 6 weeks of age. Not only sham-operated mice, but also neonatally thymectomized mice could completely eliminate the bacteria from the spleen and liver, while athymic nude mice could not eliminate them and showed a persistent form of infection. A strong delayed footpad reaction and acquired cellular resistance could be raised in neonatally thymectomized mice just as well as in sham-operated mice, but not in athymic nude mice. The delayed footpad reaction could be induced in neonatally thymectomized mice without an accompanying ability to inhibit macrophage migration. These results suggest that T cells responsible for immunity against listerial infection require the presence of the thymus for only a very short period in their development.

Immune reaction of tumor-bearing mice to Propionibacterium acnes and the antitumor effect of the bacteria.

The relationship between immunological reaction to Propionibacterium acnes (PA) and the antitumor effect of the injected bacterium was investigated. The aim was to determine whether the strength of the immune reaction to the bacterium can be used to predict its antitumor effectiveness. C3Hf/Sed mice received SC injections (right thigh) of viable cells of a methylcholanthrene-induced fibrosarcoma. When the tumor grew to 5 mm, the hosts received 350 micrograms PA IV as the antitumor treatment. Cellular immunity (footpad test) to PA was assayed in one group of these mice 14 days later, and in the other anti-PA agglutinins were determined 28 days later. The PA injection cured 22 of 58 mice in the first, and 20 of 46 mice in the second group. Footpad reaction and agglutinin titers to PA in cured mice were not statistically different from those in mice eventually killed by the tumor. Therefore, the strength of the immune reaction to PA in tumor-bearing mice could not be used to predict the antitumor effectiveness of the bacterium.

In vitro induction of suppressor T-cells in delayed-type hypersensitivity to BCG and an essential role of I-J positive accessory cells

Antigen-specific suppressor T-cells in delayed-type hypersensitivity (DTH) to BCG were induced in vitro. Normal spleen cells of C3H/He mice were incubated with 50 μg of PPD per ml for 4 days at 37°C, and the non-adherent cells in the culture were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer, and DTH was measured by the footpad reaction to PPD two weeks later. Footpad reaction to PPD was positive in CY-treated C3H/He mice immunized to BCG, while it was suppressed by the transfer of the in vitro induced suppressor cells. When the suppressor cells were treated with anti-thy-1.2 antiserum and complement before transfer, the suppression was abrogated. Next, the spleen cells were separated into plastic adherent and non-adherent fractions. After treatment with anti-thy-1.2 and complement, the adherent cells were treated with either anti-I-J k or anti-I-A k anti-serum and complement. Then, they were reconstituted with the non-adherent cells and cultured with PPD. Treatment of the adherent cells with anti-I-J k antiserum and complement abrogated the suppressor cell induction, while the treatment with anti-I-A k had no effect. These facts indicate that I-J positive non-T-adherent cells play an essential role in the induction of suppressor cells in DTH.

Direct correlation between delayed footpad reaction and resistance to local bacterial infection.

The resistance to bacteria was studied at the site of delayed footpad reaction in mice immunized with Listeria monocytogenes. When a challenge injection of listeria was given into the footpad of immune mice, no enhancement of bacterial elimination was observed before the generation of delayed footpad reactivity. After the generation of delayed reactivity, an enhanced elimination of listeria or Salmonella typhimurium was observed only at the site of strongly positive delayed footpad reaction elicited with listerial antigen. Such an enhancement in bacterial elimination was also found at the site of delayed footpad reaction induced by immunization with heterologous erythrocytes. Both delayed footpad reaction and local resistance could be transferred locally by immune spleen cells in a dose-dependent fashion, but were completely abrogated when recipient mice were treated with carrageenan or whole-body X irradiation. Macrophage accumulation was an important factor in the expression of resistance at the reaction site. These results suggest that the delayed footpad reaction contributes to host defense by enhancing the local resistance to bacteria.

Depression by Pseudomonas aeruginosa of two T-cell-mediated responses, anti-Listeria immunity and delayed-type hypersensitivity to sheep erythrocytes.

Pseudomonas aeruginosa was studied for its effects on T-cell-mediated responses in mice, as exemplified by anti-Listeria immunity and delayed-type hypersensitivity to sheep erythrocytes. Immunity to Listeria monocytogenes was measured by quantitation of bacteria in spleens and mortality; delayed hypersensitivity to sheep erythrocytes was tested by the footpad reaction. Three different preparations of P. aeruginosa were used: the supernatant of a heat-killed culture, living bacteria, and heat-killed organisms. Similar results were obtained with the three preparations. Administration of P. aeruginosa 24 h before Listeria infection reduced the resistance to the secondary challenge, as measured by increased bacterial multiplication in the spleen and rate of mortality. Cell transfer experiments showed that pretreatment of normal recipient mice with P. aeruginosa prevented them from being adoptively immunized against a Listeria challenge infection with spleen cells from immune donors. They also showed that treatment of donors with P. aeruginosa before immunization affected the capacity of their spleen cells to protect normal recipients against Listeria. Furthermore, spleen and peritoneal exudate cells obtained from mice given P. aeruginosa were capable of preventing immunization of normal recipients against Listeria. Similar results were obtained when the delayed hypersensitivity response to sheep erythrocytes was studied. The suppressive activity of P. aeruginosa-treated spleen cells was lost by removing adherent cells. Conversely, the adherent, heat-killed, anti-immunoglobulin-treated spleen cells exerted a suppressor effect. It thus appears that P. aeruginosa injection changes macrophage and T-lymphocyte activities and results in the development of adherent, macrophage-like suppressor cells in the spleen and peritoneal cavity.

Cellular regulation of lung granuloma formation and delayed-type hypersensitivity induced with BCG cell walls further evidence of involvement of cellular immunity in lung granuloma formation

We have previously showed several evidences in which cellular immunity is involved in lung granuloma formation induced with oil-associated BCG cell wall (CW) vaccine. In the present study, we examined in mice the effects of thymectomy, splenectomy and the administration of cyclophosphamide on lung granuloma formation as well as BCG CW-induced footpad reactions to PPD, a prototype of delayed-type hypersensitivity (DTH). The results presented herein provide further evidence that cellular immunoregulatory mechanisms control the development of lung granuloma and DTH, and that lung granuloma formation is thymus- and spleen-dependent.

Induction of a delayed type hypersensitivity to carrageenin in mice.

Induction of delayed type hypersensitivity to carrageenin in mice was investigated with successful results. Male ddY/s mice were immunized with 1 mg of carrageenin 100 microliters of Freund's complete adjuvant-saline (1:1) emulsion and challenged 10 d after the immunization on their footpads with either 10 or 40 micrograms of the antigen in 2.5 microliters of Freund's incomplete adjuvant-saline (1:1) emulsion. The mice exhibited a strong allergic footpad reaction at 24-48 h after the challenge injection. The footpad reaction was successfully transferred with spleen cells. Non-steroidal antiinflammatory drug, indomethacin, and steroidal antiinflammatory drug, dexamethasone, suppressed the delayed type hypersensitivity reaction.

Experimental study on immunological reactions against the articular cartilage.

In rats immunized with articular cartilage antigens, delayed hypersensitivity was detected by the macrophage migration inhibition test and delayed footpad reaction. Additionally, a proliferative response was found in the regional lymph nodes of immunized rats. The mixed addition of sensitized lymphocytes and antibodies exerted a cytotoxic effect on cultured chondrocytes. Articular damage including accelerated chondrolysis may have some relation to delayed hypersensitivity to autologous articular cartilage antigens.

Mechanisms in the in vivo release of lymphokines: relationship of high and low responsiveness to other parameters of the immune response

Variations exist between different strains of inbred mice in the release of lymphokines into the circulation. A number of manifestations of cell-mediated immunity in mice sensitized intravenously with Mycobacterium bovis BCG were analyzed to determine their association with the in vivo release of gamma interferon (IFN-gamma) and migration inhibitory factor. Differences occurred among the strains in the proliferative responses of splenic cells to specific antigen and in the release of IFN after the challenge of BCG-sensitized mice with lipopolysaccharide. However, the capacity of an individual strain to release migration inhibitory factor and IFN-gamma into the circulation did not parallel the extent either of the proliferative responses or of the release of IFN induced by lipopolysaccharide. Not all of the strains developed marked delayed footpad reactions to challenge with PPD regardless of the extent of their responses by other parameters. Delayed footpad reactions did develop in mice sensitized via the subcutaneous route, although this sensitization did not result in the capacity to release migration inhibitory factor and IFN-gamma into the circulation of individual inbred strains.