Reiko M. Nakamura

Th1 Adjuvant N-Acetyl-d-Glucosamine Polymer Up-Regulates Th1 Immunity but Down-Regulates Th2 Immunity against a Mycobacterial Protein (MPB-59) in Interleukin-10-Knockout and Wild-Type Mice

ABSTRACT Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-μm chitin particles (nonantigenic N-acetyl-d-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-γ) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mφ) (PGE2-Mφ), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mφ formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-γ-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersentivity (DTH) footpad reactions were detected. On the other hand, chitin–MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG–MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.

Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG

Hosts infected with low doses of mycobacteria develop T helper cell type 1 (Th1) immunity, but at relatively higher doses, a switch to Th2 immunity occurs. Prostaglandin E2 (PGE2) is a proposed mediator of the Th1‐to‐Th2 shift of immune responses, and mycobacterial products induce PGE2‐releasing macrophages (PGE2‐MØ) in the mouse spleen in a dose‐dependent manner. Splenic PGE2‐M Ø from Balb/c mice, given 0.01 or 1 mg heat‐killed (HK) Mycobacterium bovis bacillus Calmette‐Guerin (BCG) intraperitoneally (i.p.), were characterized by the ex vivo release of PGE2 (>10 ng/106 cells), cytokine production, and expression of PGG/H synthase (PGHS)‐1, PGHS‐2, cytosolic PGE synthase (PGES), and microsomal PGES‐1. At Day 14 after the treatment, mice treated with 1 mg, but not 0.01 mg, BCG had increased levels of PGHS‐2+ PGE2‐MØ, total serum immunoglobulin E (IgE), and serum IgG1 antibodies (Th2 responses) against heat shock protein 65 and purified protein derivative. Cultures of spleen cells isolated from these mice expressed interleukin (IL)‐4 and IL‐10 in recall responses. Treatment of mice receiving 1 mg BCG with NS‐398 (a PGHS‐2 inhibitor, 10 mg/kg i.p., daily) resulted in enhanced interferon‐γ (IFN‐γ) production with reduced IL‐4 and IL‐10 production in recall responses. This treatment also resulted in decreased total serum IgE levels. Treatment of C57Bl/6 mice with HK‐BCG (0.5 mg dose) also induced a mixture of Th1 and Th2 responses, although IFN‐γ production was markedly increased, and IL‐4 was decreased compared with Balb/c mice. Thus, our results indicate that by 14 days following treatment of mice with high doses of HK‐BCG, splenic PGE2‐MØ formation is associated with a PGHS‐2‐dependent shift from Th1‐to‐Th2 immune responses.

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 micrograms of PPD per ml for 4 days at 37 degrees 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-Jk or anti-I-Ak antiserum and complement. Then, they were reconstituted with the non-adherent cells and cultured with PPD. Treatment of the adherent cells with anti-I-Jk antiserum and complement abrogated the suppressor cell induction, while the treatment with anti-I-Ak 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.

Detection of Active Tuberculosis by an MPB-64 Transdermal Patch: A Field Study

The mycobacterial antigen MPB-64 was formulated for delivery in a transdermal patch and used as a diagnostic skin test reagent to detect active tuberculosis (TB) in patients attending a clinic in Manila, The Philippines. The MPB-64 Transdermal Patch was applied to 62 patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and to 28 non-TB but tuberculin-positive controls. The results were read at 72 h. The sensitivity of the Transdermal Patch was 87.8%, with an efficacy of 92.9% and a specificity of 100%. The 13 TB patients who had completed 6 months of TB chemotherapy showed different reactions to the MPB64 patch test: those who had completed chemotherapy < 4 months before testing were positive; 50% of patients who completed chemotherapy 5 months previously were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. Moreover, the MPB64 Transdermal Patch may be useful to monitor successful chemotherapy.The mycobacterial antigen MPB-64 was formulated for delivery in a transdermal patch and used as a diagnostic skin test reagent to detect active tuberculosis (TB) in patients attending a clinic in Manila, The Philippines. The MPB-64 Transdermal Patch was applied to 62 patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and to 28 non-TB but tuberculin-positive controls. The results were read at 72 h. The sensitivity of the Transdermal Patch was 87.8%, with an efficacy of 92.9% and a specificity of 100%. The 13 TB patients who had completed 6 months of TB chemotherapy showed different reactions to the MPB64 patch test: those who had completed chemotherapy < 4 months before testing were positive; 50% of patients who completed chemotherapy 5 months previously were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. Moreover, the MPB64 Transdermal Patch may be useful to monitor successful chemotherapy.

I-J-positive cloned macrophages as accessory cells for the induction of suppressor T cells in vitro

SummaryI-A+/I-J+ cloned macrophages, SL-1, played the role of APC in the in vitro induction of Ts against DTH to BCG. By treating SL-1 cells with various antibodies, it was shown that I-J antigens on SL-1 cells are essential for Ts induction, but not for the effector cell induction for DTH. Ia-negative cloned macrophages, SL-4, did not show any APC activity either in suppressor or effector cell induction. The precursors of the Ts were also I-J-positive, and I-J restriction resided between T cells and macrophages in the Ts induction. Thus, it is suggested that the pre-Ts recognizes the antigenic determinants of BCG presented on the APC in association with the I-J antigen and differentiates into the Ts. This pathway seems analogous to that of helper or effector T induction, where the antigenic determinant is recognized by a T cell in association with the I-A antigen on APC.

Macrophage activation for tumor cytotoxicity: control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines.

Abstract Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.

Cell-cell interaction responsible for the induction of first order suppressor T cells in hapten-specific contact sensitivity reactions

SummaryC3H/He mice were found to be low responders in the contact sensitivity response to ABA. Intravenous injection of ABA-coupled syngeneic spleen cells induced hapten-specific Ts in C3H/He mice. These cells were Ts1 because they acted on the inductive phase of the contact sensitivity. They could suppress the contact sensitivity in H-2-compatible CBA mice which were known to be high responders to ABA. Using in vivo and in vitro systems for the induction of Ts, it was shown that I-A−J-J+Thy-1− adherent cells were necessary as APC for the induction of Ts1.

Suppressor Cells in Mycobacterial Infections

Mycobacterium bovis BCG has been widely used as the antituberculosis vaccine and is also a well-known immunopotentiator in both humoral and cell-mediated immune responses. Bacillus Calmette-Guerin (BCG) or Mycobacterium tuberculosis is an indispensable component of Freund’s complete adjuvant and has been used in various experimental systems to potentiate immune responses. Because of its adjuvant activity, BCG has been used for immunotherapy of cancer, such as acute lymphoid leukemias1 or melanomas2 in humans. There are quite a few reports in this field both in animal experiments and in human cases,3–7 many of which indicated improvement or even cure of the tumor by the immunotherapy with BCG. However, several reports show enhanced tumor growth by treatment with BCG. Likhite4 reported that in some instances tumor growth was enhanced by BCG injection in rats. Piessens et al.8 showed that injection of a tumor cell-BCG mixture into rats resulted in either suppressed or enhanced growth of the tumor depending on the dose of BCG. Mathe,9 who had emphasized the immunotherapeutic effect of BCG, even found that a high dose of BCG suppresses antibody production against sheep red blood cells (SRBC) in mice. BCG injected intravenously (IV) induced suppressed immunity to rodent malaria.10 Thus, it has been known for some time that the famous immunopotentiator BCG has double-faced activity, modulating immune responses either into a positive (immunity) or negative (suppression) direction.

Non-H-2-linked difference in delayed-type hypersensitivity in mice

Abstract Significant difference in delayed-type hypersensitivity (DTH) to sheep erythrocytes (SRBC) and to Mycobacterium bovis BCG was observed between SJL/J (H-2 s ) and A.SW (H-2 s ) strains. SJL/J was a high responder in DTH to BCG but a low responder to SRBC, while A.SW mice showed a high response to SRBC but a low response to BCG. Cyclophosphamide treatment of these mice resulted in the enhanced DTH to the antigen in animals which were in low responsive state, while being unable to enhance the response of high responders. These results suggest that DTH to either SRBC or BCG is regulated by a gene(s) which is not linked to H-2, and that the low responsiveness is probably due to a suppressor mechanism.

Accessory Cell Function of a Lined Macrophage in the Induction of Suppressor T Cells in vitro

Antigen-specific Ts against DTH to BCG were induced in vitro by culturing the splenic T cells of C3H/He mice together with the lined macrophage, SL-1, pulsed with PPD. The suppressor activity was determined by the suppression of DTH in CY-treated syngeneic recipients which had received the cultured cells before BCG sensitization. SL-1 cells express I-A and I-J antigens on the surface, while SL-4 cells, which originated in the same mouse strain as SL-1, do not express any Ia antigens. When I-A antigens on SL-1 were blocked by anti-I-A antibody without complement before PPD-pulsing, the induction of Ts was rather enhanced. When I-J antigens on SL-1 were blocked by anti-I-J alloantiserum, no Ts were induced. Instead, these I-J-blocked and PPD-pulsed SL-1 cells induced effector cells of DTH in vitro. Ia-negative lined macrophage SL-4 cells did not show any activity either in suppressor or in effector cell induction. From these results, we propose a model for the immune modulation led by APC.