Sensitized Spleen Cells Research Articles

Investigations on the anti-inflammatory and anti-allergic activities of the leaves of Uncaria guianensis (Aublet) J. F. Gmelin

Uncaria guianensis (Aublet) J. F. Gmelin is an herbal medicine from tropical areas of South and Central America. We investigated the anti-inflammatory and anti-allergic properties of an ethanolic extract of U. guianensis leaves, containing alkaloids, flavonoids and phenol carboxylic acids, as revealed by thin layer chromatography (TLC). Oral pre-treatment with U. guianensis inhibited zymosan-induced paw oedema (500 mg/paw) and pleural exudation (100 mg/kg) within 4 h (25-200 mg/kg). U. guianensis (100 mg/kg) inhibited total leukocyte and neutrophil numbers in the pleural cavity 4 h after zymosan stimulation. Pre-treatment with U. guianensis (100 mg/kg, p.o.) inhibited total leukocyte, neutrophil and eosinophil recruitment into the pleural cavity 24 h after LPS (250 ng/cavity, i.t.). Pre-treatment with U. guianensis inhibited paw oedema (25-200 mg/kg) induced by ovalbumin (OVA) within 1 h, and neutrophil and eosinophil recruitment into the mice pleural cavity 24 h after OVA (100 mg/kg). In vitro data revealed that U. guianensis impaired LPS-induced nitric oxide and CXCL8 generation by murine peritoneal macrophages, as well as OVA-induced interleukin-5 synthesis by previously sensitized spleen cells. These results demonstrate that U. guianensis leaves provide effective anti-inflammatory and anti-allergic activities.

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL(+)/B7-1(+)SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P<0.01). SGC-7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC-7901 cells (z = 2.06-44.30, P<0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1+/-2.4% vs 30.5+/-2.3%, P<0.05). FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

Transforming growth factor-beta has contrasting effects in the presence or absence of exogenous interleukin-12 on the in vitro activation of cells that transfer experimental autoimmune thyroiditis.

Mouse thyroglobulin (MuTg)-sensitized spleen cells activated in vitro with MuTg induce experimental autoimmune thyroiditis (EAT) in recipient mice with a thyroid infiltrate consisting primarily of lymphocytes. A more severe and histologically distinct granulomatous form of EAT (G-EAT) is induced when donor cells are activated with MuTg together with anti-interferon-gamma (IFN-gamma), anti-interleukin-2 receptor (IL-2R) monoclonal antibody (mAb), and IL-12. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that can both suppress and exacerbate autoimmune diseases and often has inhibitory effects on lymphocytes. To determine if TGF-beta could modulate the in vitro activation of effector cells for G-EAT, TGF-beta was added to cultures of MuTg-sensitized donor spleen cells together with MuTg. Cells activated in the presence of 2 ng/ml TGF-beta induced moderately severe G-EAT in recipient mice. G-EAT induced by cells activated in the presence of TGF-beta was histologically similar but less severe than the G-EAT induced by cells activated in the presence of IL-12. IL-12 and TGF-beta modulate the activation of G-EAT effector cells by distinct mechanisms, as cells activated by TGF-beta could induce G-EAT in the presence of anti-IL-12, and TGF-beta inhibited the effects of IL-12 on EAT effector cells. TGF-beta exerted its activity during the first 24 h of the 72-h culture, whereas IL-12 functioned primarily during the final 24 h of culture. These results indicate that thyroid lesions with granulomatous histopathology can be induced by both IL-12-dependent and IL-12-independent mechanisms, and TGF-beta can exert both positive and negative effects on the effector cells for G-EAT.

Induction of experimental autoimmune thyroiditis in IL-12-/- mice.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg and anti-IL-2R or MTg and IL-12. Previous work suggested that IL-12 was required in vitro for development of G-EAT. To determine whether IL-12 was also required during the induction and/or effector phases, DBA/1 mice with a disrupted IL-12-P40 gene (IL-12(-/-)) were used for EAT induction. Cells from MTg-sensitized IL12(-/-) donors activated in vitro by MTg or MTg and anti-IL2R induced severe EAT in recipient mice. Compared with effector cells from IL-12(+/+) donors, effector cells from IL-12(-/-) donors induced thyroid lesions dominated by lymphocytes with minimal granulomatous changes. Thyroids of recipients of IL-12(-/-) cells expressed less IFN-gamma mRNA and more TGF-beta, IL-4, and IL-10 compared with recipients of IL-12(+/+) cells. When IL-12 was added during in vitro activation, cells from both IL-12(-/-) and IL-12(+/+) donors induced severe G-EAT, and expression of all cytokines except IL-12 was comparable in thyroids of both IL-12(+/+) and IL-12(-/-) recipients. Transfer of cells from IL-12(+/+) or IL-12(-/-) donors into IL-12(+/+) or IL-12(-/-) recipients indicated that IL-12 expressed in thyroids was derived from recipients. Thus, endogenous IL-12 is not absolutely essential for the sensitization and activation of EAT effector cells to induce severe EAT, although it is required in vitro to promote activation of cells to induce severe granulomatous histopathology.

Effect of Sensitized Macrophages in Transplantable Sarcoma in Mice: Synergistic Effect of Hyperimmunized Serum, Sensitized Spleen Cells and Macrophages in Tumor Transplantation

Background Immunosuppression cannot develop tumors by itself. It may induce tumors under appropriate conditions and may accelerate tumor development which may be reversed by sensitized spleen cells. This study concerns the effect of sensitized macrophages in murine-transplantable sarcomas by a combination of hyperimmune serum, sensitized spleen cells and macrophages. Methods The main technique adopted was intraperitoneal (ip) injection of 2 mL of 10% protease peptone broth, followed 2 days later by inoculation into the peritoneum with 10-mL Hank isotonic solution. The cells from the pooled peritoneal fluid were tested by dye exclusion test to ascertain the percentage of live cells. They were tried in whole body-irradiated mice with a combination of immune serum and sensitized spleen cells to ascertain whether a suppression of growth of solid tumors could be achieved when subcutaneously (sc) administered with the previously mentioned combinations. Results The addition of immunomacrophages from transplanted tumor-bearing mice significantly suppressed the growth of subcutaneous solid tumors when the number of tumor cells was kept constant. A change in number of immunomacrophages from hyperimmunized mice at a ratio of 4:l showed a direct relationship in suppression of tumor growth. Experiments were initiated in which tumor cells were injected sc and peritoneal macrophages were injected either intravenously (iv) or ip. Experiments were then initiated to prove that cell-to-cell contact is essential for tumor suppression. In experiments in which tumor cells were administered sc and macrophages injected either iv or ip, a significant immunosuppressive effect was not shown, thus also indicating that regardless of which, cell-to-cell contact is an absolutely essential factor involved in tumor suppression. A combination of hyperimmune serum and macrophages was found to act synergistically. Macrophages and hyperimmune serum at a lesser proportion did not suppress tumor growth. Sensitized macrophages and spleen cells together significantly suppressed tumor growth in a pure isogenic strain of irradiated mice. The sensitized macrophages injected iv prolonged the survival period and retarded tumor growth. Conclusions Tumor suppression by macrophages was found to be due to its contact with tumor cells that enables the effective transfer of immunity. Hyperimmune serum and other cells (macrophages, spleen lymphocytes) act synergistically toward each other and prolong the survival period.

Suppressive effects of co-stimulatory molecule expressions on mouse splenocytes by anti-allergic agents in vitro.

The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro.

A Thyroxine-containing Thyroglobulin Peptide Induces both Lymphocytic and Granulomatous Forms of Experimental Autoimmune Thyroiditis

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg can induce two histologically distinct forms of experimental autoimmune thyroiditis (EAT). MTg-sensitized cells activated with MTg alone induce a mild chronic form of EAT in which the thyroid infiltrate consists primarily of lymphocytes and other mononuclear cells (lymphocytic EAT). The same donor cells activated with MTg and anti-IL2R mAb induce a more severe and acute form of EAT with a thyroid inflammatory lesion having granulomatous histopathological features. A thyroxine-containing (T4) peptide, corresponding to positions 2549–2560 of human Tg, was shown by others to activate spleen cells of mouse thyroglobulin (MTg)-sensitized CBA/J mice to induce lymphocytic EAT. To determine if the CD4+effector T cells that induce granulomatous EAT can respond to the same T-cell epitope, the present study was undertaken to determine if both forms of EAT could be induced by the 2549–2560 thyroxine (T4)-containing peptide. This peptide was very effective for activation of T cells from MTg-primed CBA/J donors to induce granulomatous EAT but, in contrast to MTg, did not activate T cells from AKR/J or DBA/1 mice to induce granulomatous EAT. The T4 peptide did not apparently activate peptide-specific B cells in vivo but did activate MTg-primed B cells in vitro to produce anti-MTg autoantibody in recipient mice. These results demonstrate that a single 12-amino-acid thyroxine-containing peptide can activate T cells from CBA/J mice to induce both lymphocytic and granulo-matous EAT. However, this peptide does not activate T cells from some other EAT-susceptible strains of mice, suggesting that MTg contains multiple epitopes able to activate T cells to induce granulomatous EAT.

Experimental Hepatitis in Neonatally Thymectomized Mice: Transfer of Disease and the Role of T Cells

Using neonatally A/J thymectomized mice, we have produced chronic hepatitis by administration of sublethal doses ofPropionibacterium acnesand lipopolysaccharide (LPS). Our goal in this unique model was to evaluate the effector cell population required to generate chronic hepatitis by transferring spleen cells or splenic subpopulations derived from donor thymectomized mice with chronic hepatitis into congenic recipient nonimmunized thymectomized or sham-thymectomized animals. Several key observations were made regarding the ability to induce and to transfer disease. First, an inflammatory liver injury in neonatally thymectomized (NTx) mice was readily generated using sublethal doses ofP. acnesand LPS. Second, the lesions were persistent and associated with the production of autoantibodies to liver-specific lipoprotein and anti-nuclear antibodies. Third, these features were not found in comparably injected nonthymectomized control A/J mice. Fourth, the same liver injury was transferred to neonatally thymectomized but otherwise naive mice by the transfer of donor spleen cells from affected mice previously induced to develop experimental hepatitis. Fifth, the transfer of this liver injury could not be achieved using T-cell-depleted spleen cells. Deletion of CD4+T cells or CD8+T cells by sensitized spleen cells resulted in suppression of the transferred liver injury. In contrast, transfer of nylon wool adherent splenic T cells induced severe hepatitis. These data suggest that the chronic liver injury induced in NTx mice by administration ofP. acnesand LPS involves a breakdown in tolerance accompanied by the appearance of autoantibodies and that nylon wool adherent CD4+and CD8+T cells play important roles in the modulation of liver injury.

T-T cellular interaction between CD4-CD8- regulatory T cells and T cell clones presenting TCR peptide. Its implication for TCR vaccination against experimental autoimmune encephalomyelitis.

Regulatory T cells recognizing TCR determinants presumably play a critical role in the control of experimental autoimmune encephalomyelitis, a prototype tissue-specific autoimmune disease. This study was initiated to determine whether regulatory T cells can be induced against a V beta 17a CDR2 peptide (residues 50-68) in SJL/J mice. Although the TCR peptide showed regulatory effects in vivo, the presence of T cells specific for the peptide could not be proven with conventional proliferation assays. Unexpectedly, in the presence of myelin basic protein-specific T clone cells (Tcc), the sensitized spleen cells vigorously proliferated in response to the TCR peptide. The subsequent experiment showed that this was due to the outstanding capability of the Tcc as APC for the exogenous TCR peptide. Using the Tcc as APC, we were able to establish V beta 17a50-68-specific T cell lines from in vivo primed spleen cells. The line cells were MHC class I restricted and dominated by T cells with a distinct surface phenotype (CD4-CD8-V beta 17a+). Presentation of the peptide by the Tcc was inhibited by treatment with gelonin that could block a MHC class I presentation pathway. The ability of T cells to present the TCR peptide was not related to their Ag specificity, but correlated with the expression levels of MHC class I molecules and adhesion molecules such as intercellular adhesion molecule-1 and B7-1 on their surface. The TCR peptide-specific T cells produced a soluble mediator(s) that is inhibitory for T cell activation and were protective against actively induced experimental autoimmune encephalomyelitis. These results show that V beta 17a50-68 vaccination induces regulatory CD4-CD8- T cells that could interact with T cells presenting relevant TCR fragments.

T Cell Receptor Vβ Usage in Murine Experimental Autoimmune Thyroiditis

Mouse thyroglobulin (MTg)-sensitized spleen cells activatedin vitrowith MTg induce experimental autoimmune thyroiditis (EAT) in which the thyroid infiltrate consists primarily of mononuclear cells (MNC) (lymphocytic EAT). MTg-sensitized spleen cells cultured with MTg together with anti-IL2R antibody induce a granulomatous form of EAT in which the thyroid is infiltrated by MNC in addition to PMNs, histiocytes, and multinucleated giant cells. CD4+T cells are the primary effector cells for both forms of EAT. The presence of specific T cell receptor (TCR) Vβ families in the thyroid infiltrate was examined by flow cytometry and reverse transcription–polymerase chain reaction (RT–PCR) amplification of mRNA. At the time of maximal disease severity, cells infiltrating the thyroid expressed primarily Vβ8, Vβ4, Vβ11, and Vβ14 as determined by flow cytometry. RT–PCR confirmed these findings and also detected several additional Vβ gene families, including Vβ1, Vβ2, Vβ6, Vβ13, and Vβ15; Vβ3, Vβ10, and Vβ12 were also detected in some, but not all, experiments. There were no differences in the Vβ T cell repertoires in thyroids of mice with lymphocytic vs granulomatous EAT. RT–PCR analysis of intrathyroidal MNC 11 days after cell transfer showed TCR Vβ mRNA transcripts to be primarily restricted to Vβ4, Vβ11, and Vβ14, whereas the predominant thyroid-infiltrating T cell 21 days after cell transfer was Vβ8+. Depletion of Vβ8+T cells in recipient mice did not reduce EAT severity. TCR Vβ usage shifted predominantly to Vβ4+, Vβ11+, or Vβ14+T cells of both CD4+and CD8+T cell subsets. These results indicate that multiple Vβ TCR are expressed in thyroids of mice with EAT.

The role of alpha 4 integrin and intercellular adhesion molecule-1 (ICAM-1) in murine experimental autoimmune thyroiditis.

Mouse-thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg induce a lymphocytic form of experimental autoimmune thyroiditis (EAT) whereas activation of the same cell population with MTg in the presence of anti-interleukin 2 receptor antibody (M7/20) induces a granulomatous form of EAT. The thyroid infiltrate in both lymphocytic and granulomatous EAT includes both CD4+ and CD8+ T cells and CD4+ T cells are the primary effector cells for both forms of EAT. This investigation was undertaken to begin to define the roles of alpha 4 integrin, and intercellular adhesion molecule-1 (ICAM-1) in the migration of CD4+ and CD8+ T cells to the thyroid in EAT. The studies presented here demonstrate the expression of alpha 4 integrin and ICAM-1 on CD4+ and CD8+ T cells infiltrating the thyroid and the expression of vascular cell adhesion molecule (VCAM) and ICAM-1 on thyroid cells of mice with EAT. The effects of anti-alpha 4 and anti-ICAM mAb administration on EAT severity in recipient mice was also determined. Anti-alpha 4 administration reduced or abolished lymphocyte infiltration in the thyroid resulting in reduced severity of both lymphocytic and granulomatous EAT. In contrast, anti-ICAM mAb had little effect on EAT severity. These results suggest that these two adhesion molecules exhibit differential functional roles in the modulation of EAT disease severity and that alpha 4-VCAM interactions may be of particular importance in trafficking of effector cells to the thyroid.

Adoptive transfer of experimental hypersensitivity pneumonitis in the LEW rat.

The pathogenesis of immune-mediated pulmonary inflammation following inhalation of antigen in sensitized subjects may involve specific cellular or humoral mechanisms, or both. We used adoptive transfer to document cellular mechanisms in an established LEW rat model of acute hypersensitivity pneumonitis. The optimal protocol utilized sensitized spleen cells cultured for 72 h in the presence of antigen and concanavalin A before intraperitoneal injection of 20 x 10(6) cultured cells into sublethally irradiated recipients. Experimental and control rats were subjected 7 d later to twice daily aerosol challenge for 1 or 3 d and examined 12 h after the last challenge. Histopathology was evaluated in one lung, and the other was lavaged and cells evaluated by flow cytometry for T cell phenotypes (CD4, CD8, and RT6, CD45R subsets). Histopathology and cell numbers for most phenotypes were increased in experimental over controls, but proportions were unchanged. Similar results were obtained in animals evaluated at both 24 and 72 h after initial challenge. Results indicate cellular participation in an established LEW rat model of HP. Significant increases in numbers of T cell phenotypes suggest an important role in pathogenesis, but no single phenotype could be incriminated by disproportionate numbers.

Regulation of the Induction and Resolution of Granulomatous Experimental Autoimmune Thyroiditis in Mice by CD8 + T Cells

Mouse thyroglobulin (MTg)—sensitized spleen cells activated in vitro with MTg induce experimental autoimmune thyroiditis (EAT) in which the thyroid cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Cells cultured with MTg plus anti-IL2R antibody induce EAT having a granulomatous histopathology. CD4 + T cells are required to induce both forms of EAT. The potential contribution of CD8 + T cells in the pathology of lymphocytic vs granulomatous EAT was addressed using anti-CD8 mAb to deplete CD8 + T cells from donor or recipient mice. Depletion of donor CD8 + T cells had little effect on EAT severity induced by cells cultured with MTg or MTg plus anti-IL2R mAb. However, recipients of CD8-depleted cells activated with MTg induced granulomatous EAT whereas cells from untreated donors activated with MTg alone induced lymphocytic EAT. Similar results were obtained when anti-CD8 mAb was added to cultures to block activation of CD8 + T cells. Injection of anti-CD8 mAb into recipient mice slightly increased the severity of EAT induced by cells cultured with MTg or MTg and anti-IL2R mAb when EAT was assessed 19-20 days after cell transfer. Cells cultured with MTg alone induced mild granulomatous EAT in most anti-CD8-treated recipients. When thyroids of mice which had granulomatous EAT on Day 19 were examined 47 or 60 days alter cell transfer, the granulomatous inflammatory response in untreated recipients had almost completely resolved. In contrast, granulomatous EAT persisted and often became more severe in anti-CD8-treated recipients. Anti-CD8 treatment did not influence lymphocytic EAT assessed late after cell transfer. These results indicate that CD8 + T cells are required for the resolution of granulomatous EAT.

Chronological changes in autoantigenicity of autologous testicular germ cells in C3H/He mice during the postnatal period

Our recent studies demonstrated that experimental autoimmune orchitis (EAO) model was produced in C3H/He mice with high incidence by two subcutaneous injections of viable syngeneic testicular germ cells (TC) without the use of any adjuvants or immunopotentiators. In this study the developmental patterns of autoantigenicity of TC during postnatal period were investigated by examining the orchitogenic activity of TC, the lymphostimulatory activities of TC (including the TC-induced in vitro lymphocyte proliferative response and the cytokine release from sensitized spleen cells (SPC) in response to TC) and the immunohistochemical localization of target autoantigens in the testes of mice at various weeks of age. Delayed-type hypersensitivity-inducing capacity and anti-TC antibody-eliciting capacity were initially observed in mice that were immunized with TC of 4-week old (w.o.) mice. The TC from 6-w.o. mice had the capability of inducing EAO (orchitogenicity) for the first time. A significant stimulation of in vitro lymphocyte proliferative response, as well as of interleukin (IL) 5 and IL-6 production by sensitized SPC were detectable when TC of mice 3-w.o. or more than were employed as stimulant. IL-2 and interferon gamma production were detected with TC of 4-w.o. mice. Immunohistochemical staining reaction with anti-TC antisera was primarily localized at the acrosomal portion of spermatids and spermatozoa in the seminiferous tubules, being already detected in spermatids of as early as 3-w.o. mice. Thus, from these data it is suggested that the appearance of the lymphostimulatory activities of TC consistently precedes that of the orchitogenic activity and that relatively mature germ cells such as spermatids and spermatozoa developing in the testes during the postnatal weeks may be responsible for the induction of disease and relevant immune responses in our EAO system.

Differential requirement for autoantibody-producing B cells for induction of lymphocytic versus granulomatous experimental autoimmune thyroiditis.

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTG transfer experimental autoimmune thyroiditis (EAT) in which the thyroid cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Addition of anti-IL2R antibody to cultures with MTg leads to activation of cells that induce granulomatous EAT, accompanied by high serum anti-MTg autoantibody responses, in recipient mice. CD4+ T cells are required to induce both forms of EAT; whether B cells and/or autoantibodies produced by MTg-sensitized B cells also contribute to disease severity or the type of thyroid histopathology is unknown. In our study, B cells and autoantibody responses produced in recipient mice were reduced either by column removal of B cells from donor spleen cells or by treatment of recipient mice with anti-I-AK mAb at the time of cell transfer. These maneuvers only slightly reduced the severity of lymphocytic EAT but markedly reduced the severity and incidence of granulomatous EAT developing in recipient mice. Delaying the initiation of anti-I-AK treatment until 6 days after cell transfer was less effective in reducing anti-MTg autoantibody responses or granulomatous EAT. These studies all suggested that anti-MTg autoantibodies were required for development of granulomatous but not lymphocytic EAT. However anti-I-AK-treated recipients receiving injections of anti-MTg antibody or having serum antibody induced by prior immunization with MTg and LPS also developed less severe granulomatous EAT than controls. These results suggest that sensitized CD4+ T cells and circulating anti-MTg autoantibody are not sufficient for development of granulomatous thyroid lesions. It is possible that antibodies having a unique function or specificity are produced in mice developing granulomatous EAT or thyroid-infiltrating B cells may directly contribute to the granulomatous inflammatory response.

Murine Experimental Autoimmune Orchitis (EAO) Induced by Syngeneic Testicular Germ Cells (TC) Alone: Orchitogenic and Lymphostimulatory Activities of TC from Mice at Various Ages

As part of our continuous investigations on the immune pathogenesis of a new experimental autoimmune orchitis (EAO) model without the use of adjavant in C3H/He mice, the developmental patterns of autoantigenicity of viable syngeneic testicular germ cells (TC) during the postnatal period were investigated by examining the orchitogenic and immunogenic activities of TC, the lymphostimulatory activities of TC (the cytokine release from sensitized spleen cells (SPC) in response to TC), and the immunohistochemical localization of autoantigens in the testes of normal mice at various weeks of age. TC from 6-week-old mice had the capability of inducing EAO (orchitogenicity) for the first time. Delayed-type hypersensitivity-inducing capacity and antiTC antibody-eliciting capacity were initially observed in mice immunized with TC from 4-week-old mice. A significant stimulation of interleukin 5 (IL-5) and interleukin 6 (IL-6) production by sensitized SPC was detectable when the TC from 3-week or older mice were employed as a stimulant. Interleukin 2 (IL-2) and interferon-γ (IFN-γ) production was detected with TC from 4-week or older mice. Immunohistochemical staining reaction with anti-TC antisera was primarily localized at the acrosomal portion of spermatids and spermatozoa in the seminiferous tubules and was detected in early and late spermatids of mice as early as 3 weeks. Thus, from these chronological data it is suggested that the appearance of the lymphostimulatory activities of TC consistently precedes that of the orchitogenic activity and that the production of IL-5 and IL-6 (Th 2 cytokine) by sensitized SPC upon in vitro TC stimulation is detectable earlier than the production of IL-2 and IFN-γ (Th 1 cytokine) in the postnatal period.

Abrogation of secondary skin allograft rejection by veto-like cells in donor bone marrow.

To investigate whether a veto cell phenomenon is involved in the ability of donor strain bone marrow cells (BMC) to inactivate second-set skin graft rejection induced by the adoptive transfer of sensitized spleen cells (SSC), mice were grafted with ear skin allografts and injected on the following day with recipient strain SSC or SSC plus donor strain BMC. When skin and BMC donors from an F1 strain (B6AF1) were combined with a parental strain (C57BL/6) as recipient and SSC donors, the BMC were fully active in abrogating second-set rejection even though the SSC were naturally tolerant of the BMC. Thus, immunological recognition of the SSC by the BMC is not necessary for this abrogation. The suppressive activity of the BMC is specific in a manner similar to that reported for veto cells (i.e., they suppress an anti-self response). If BMC from various strains of mice were used to alter the rejection of C3H grafts by B6AF1 recipients, only BMC expressing the MHC antigens to which the SSC had been sensitized significantly suppressed the activity of SSC. Sharing of additional antigens between the graft and BMC (e.g., MHC antigens not recognized by the SSC or minor antigens) was neither necessary nor sufficient. Mitomycin C-treated BMC were unable to inhibit SSC-induced accelerated rejection. Overall, the characteristics of the SSC-suppressive activity of BMC are consistent with a veto cell nature of the BMC.

Abrogation of tumor-inhibitory MRC-OX8+ (CD8+) effector T-cell generation in rats by selective depletion of neutrophils in vivo using a monoclonal antibody.

These studies were designed to examine the role of neutrophils in transplantation immunity to syngeneic rat tumors. We have earlier reported that specific transplantation immunity to syngeneic transplanted tumors is abrogated by selective depletion of peripheral-blood neutrophils by administration of a monoclonal antibody (MAb) (RP-3) at the time of immunization with X-irradiated tumor cells. In order to elucidate the mechanisms of this phenomenon, we have now examined whether induction of the sensitized spleen cells that inhibit the growth of tumor cells is inhibited by RP-3 treatment at the time of in vivo priming with tumor-associated antigen (TAA). Neutrophils were required to induce the sensitized T cells responsible for tumor inhibition with Winn's assay. In addition, CD8+ T cells are proved to be effector cells in an assay system of tumor-growth inhibition using a diffusion chamber. Our results indicate that neutrophils are required for priming rats with TAA to induce CD8+ effector T cells in tumor inhibition.

Experimental murine model for autoimmune myocarditis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Experimental autoimmune myocarditis could be produced in mice by repeated injection of syngeneic heart extract together with Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) as a powerful adjuvant. Histological changes in the cardiac lesions were characterized by infiltration with mononuclear cells in the myocardium, degeneration and loss of myocardial fibers, and replacement of granulation tissues. No such cardiac lesions were produced in mice receiving injections of heart extract alone or KO3 LPS alone. Development of the autoantibody and the delayed type-hypersensitivity (DTH) against syngeneic heart extract was found in mice immunized repeatedly with the mixture of heart extract and KO3 LPS. Moreover, definite cardiac lesions were produced in normal recipient mice by transfer of sensitized spleen cells from hyperimmunized mice. Therefore, it was suggested that those cardiac lesions were caused by the autoimmune mechanism. Our methodology provided a new experimental murine model for autoimmune myocarditis.

Neurotropin inhibits experimental allergic Encephalomyelitis (EAE) in Lewis rats

The effects of Neurotropin, a substance extracted from the inflammatory dermis of rabbits inoculated with Vaccinia virus, for experimental allergic encephalomyelitis (EAE) in Lewis rats, a model for human multiple sclerosis (MS), was studied. The peptide defined by residues 68–84 (MB 68–84) which corresponds to the encephalitogenic portion of the guinea pig myelin basic protein (MBP) in complete adjuvant H37Ra (CFA) was injected into the hind foot pad of each rat. Neurotropin significantly suppressed the clinical and histological expression of actively induced EAE when administered i.p. daily from day 0 to day 6 after immunization. In addition, passive EAE induced by precultured spleen cells from rats immunized with MB 68–84 in CFA was also suppressed by daily administration of Neurotropin after cell transfer. Neurotropin treatment significantly suppressed the delayed-type hypersensitivity (DTH) response to MB 68 –84. Furthermore, the ability of spleen cells from Neurotropin-treated rats to transfer EAE was significantly lower than that of saline-treated rats. It seemed that the suppresion may be due to the inhibition of the activation by MB 68–84 of sensitized spleen cells, as demonstrated by proliferative response to MB 68–84. However, no difference was observed in Con A-induced proliferative response of the spleen cells between Neurotropin- and saline-treated rats. These findings indicate that Neurotropin inhibits EAE by suppressing the immune responses to encephalitogenic MBP with little non-specific suppression.