Role of microglia in inflammation-mediated degeneration of dopaminergic neurons: neuroprotective effect of interleukin 10.

Inflammation in the brain has been recognized to play an increasingly important role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Inflammation-mediated neurodegeneration involves activation of the brain's resident immune cells, the microglia, which produce proinflammatory and neurotoxic factors including cytokines, reactive oxygen species (ROS), nitric oxide, and eicosanoids that directly or indirectly cause neurodegeneration. In this study, we report that IL-10, an immunosuppressive cytokine, reduced the inflammation-mediated degeneration of dopaminergic (DA) neurons through the inhibition of microglial activation. Pretreatment of rat mesencephalic neuronglia cultures with IL-10 significantly attenuated the lipopolysaccharide (LPS) induced DA neuronal degeneration. The neuroprotective effect of IL-10 was attributed to inhibition of LPS-stimulated microglial activation. IL-10 significantly inhibited the microglial production of tumor necrosis factor alpha (TNF-alpha), nitric oxide, ROS and superoxide free radicals after LPS stimulation.

Loss of resistance to a highly immunogenic tumor with age corresponds to the decline of CD8 T cell activity.

A number of ultraviolet light-induced tumors that do not grow progressively in 3- or 9-month-old animals grow progressively in animals starting at approximately 15 months of age. We investigated the immune response to one such tumor, 6130, in young and old mice to determine in nature of this breakdown in immune protection with age. The 6130 tumor cells that grow progressively in 22-month-old animals still retain sensitivity of cytotoxic T lymphocytes (CTLs) generated against the highly immunogenic "6130-A" antigen and consequently are still immunologically rejected when transplanted into 3- and 9-month-old animals, but not in mice older than 15 months. Investigation of the CD8+ CTL response found that in animals 3-12 months old, primary and secondary tumor-specific CTL responses to 6130 can be generated in vivo and in vitro. On the other hand, the CD8+ CTL responses to 6130 seen in animals 15 months of age or older were significantly depressed or absent. These results indicate that a decline in antigen-specific CD8+ CTL is the major cause of increased susceptibility to 6130 tumor growth in older animals.

Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice.

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Marking hypoxic cells for complement and cytotoxic T lymphocyte-mediated lysis: using pimonidazole.

Artificial antigens are created when 2-nitroimidazoles bind to hypoxic cells. These antigens have been used in the immunodetection of tumour hypoxia but they might also serve to stimulate immune lysis of hypoxic tumour cells by complement- and cell-mediated processes. In order to test this hypothesis, lymphocytes isolated from the spleens of C3H/HeN mice that had been immunised with pimonidazole-labelled 3152-PRO cells were subcultured and tested for their ability to lyse chromium-51 loaded, pimonidazole-labelled 3152-PRO cells in an in vitro assay. In a parallel study, commercially available, rabbit complement was tested for its ability to lyse pimonidazole-labelled V79-4 cells in the presence of monoclonal antibodies which recognise protein adducts of reductively activated pimonidazole. Complement-mediated cell lysis was measured by means of an MTT assay. Complement-mediated and cell-mediated lysis was observed at pimonidazole concentrations which, in themselves, do not produce cell killing.

Identification of murine protective epitopes on the Porphyromonas gingivalis fimbrillin molecule.

Fimbriae from Porphyromonas gingivalis are believed to play an important role in the pathogenesis of periodontal diseases. The aim of the present study was to identify the fimbrial protective T-cell epitopes in CBA/J mice. A truncated protein corresponding to amino acids 1 to 198, PgF1-198, was generated and allowed us to demonstrate that the N terminus of the protein contains T-cell epitopes. With synthetic peptides, an immunodominant sequence was identified between amino acids 103 and 122. The corresponding peptide, PgF-P8, induced T-cell proliferation after in vitro restimulation of in vivo-primed cells, giving a stimulation index comparable to the one obtained with r-fimbrillin, and induced production of both Th1 and Th2 cytokines. Growth supernatant contained significant levels of interleukin 2 (IL-2), gamma interferon, IL-4 (28 pg/ml), and tumor necrosis factor alpha. Immunization of mice with r-fimbrillin, PgF1-198, and PgF-P8 induced production of antibodies specific to r-fimbrillin and PgF-P8. In addition, by using the mouse chamber model we found that mice immunized with PgF-P8 were dramatically protected against a normally lethal injection of P. gingivalis. Animals immunized with PgF-P8 40 days prior to challenge showed a 60% survival rate when challenged with P. gingivalis, compared with just 25% survival in control animals and just 5% survival in mice immunized with PgF-P8 only 21 days prior to challenge. Although the protection depended on the time of immunization before the bacterial challenge, it did not correlate with in vivo local cytokine production (IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon), specific antibody levels, or the isotype of anti-PgF-P8 antibodies produced.

Inhibition of HIV-1 by modification of a host membrane protease.

While it is clear that CD4 is the receptor for the gp120 envelope protein of HIV-1, substantial evidence suggests that other host cell proteins are required for successful membrane fusion. Studies were initiated to examine the potential for a protein receptor which has an elastase-like character to participate in fusion of HIV-1 with permissive host cells. A synthetic elastase inhibitor was shown to significantly reduce HIV-1 infectivity when present during, but not after, the initial contact between virus and cells. A human T cell elastase-like membrane component was purified and shown to be lipid-associated. By competitive inhibition, the purified protein was shown to bind gp160 within the HIV-1 fusion domain. The binding parameters of whole T cell membrane extract, with a hydrophobic pentapeptide representative of the fusion domain, suggested an elastase-like protein is the single, secondary T cell receptor for HIV-1 (K = 1 x 10(3) M-1). The pentapeptide interacted with porcine and human (epithelial and polymorphonuclear leukocyte), but not murine, elastase isoforms, suggesting its participation in the permissiveness of host cells to infection.

Generation of anti-hapten T cell cytotoxicity in vivo. Relationship to contact sensitivity and the role of contrasuppression.

Immunization procedures that induce contact sensitivity to the trinitrophenyl (TNP) hapten in vivo were investigated for their ability to induce TNP-specific cytotoxic T lymphocytes in vivo. Spleen cells from C3H/HeN mice primed for CS responses either by the topical application of picryl chloride or by the adoptive transfer of PCL immune cells show little or no cytolytic activity in vitro against TNP-coupled target cells. Intravenous immunization with TNP-substituted syngeneic spleen cells, a procedure known to make animals unresponsive to agents normally inducing CS, also failed to induce cytolytic activity in spleen cells. However, both PCL sensitization and adoptive transfer, when combined with the injection of TNP-substituted syngeneic spleen cells, induce significant cytolytic activity against TNP-haptenated BW5147 target cells in vitro. Furthermore, i.v. injection of TNP-spleen cells with surface-bound immune complexes of the IgM or IgG1 isotypes, or with a monoclonal TNP-specific contrasuppressor T cell factor also induces strong antigen-specific cytolytic activity against TNP modified targets. TcsF bears serological determinants of T cell receptor alpha and beta chains and adheres to specific antigen columns. All these immunization regimens were shown to induce CS to TNP as well as the generation of contrasuppressor T cells. The CTL generated in the spleens of immunized mice are Thy1+ CD8+ T cells an are antigen-specific and genetically restricted. The implications of these results with respect to the mechanisms by which cytolytic responses are controlled in vivo is discussed.

Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.

Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.

T cell antigen receptor immune complexes demonstrating biologic and proteolytic activity.

An elastase-like protease, recently recognized as a specific product of murine T cells, may functionally associate with the classical TCR. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes. T cell elastase and alpha 1-PI are found chemically associated with the TCR in an antigen-specific complex reminiscent of the Ig-complement 'immune complexes'.

Immunological signals which control T cell responses.

A number of identifiable immunological parameters can influence the elicitation and regulation of antigen-specific inflammatory responses to immunogenic epitopes. Injection of antigen in vivo can lead to the activation of type IV hypersensitivity responses, or to the induction of immunological tolerance to that antigen. We have used the hapten trinitrophenol as a model system for studying the factors which influence the generation and regulation of hypersensitivity responses to immunogenic epitopes in vivo. The generation of hypersensitivity or tolerance to trinitrophenyl depends on a number of immunological factors, including the form of the antigen, the route of immunization, and the presence of immune complexes of antibody and antigen on the surface of the antigen-presenting cell. Immunization with trinitrophenyl resulting in unresponsiveness can be the result of either the inability to prime inflammatory cells in vivo or the induction of suppressor T cells.

Elastase is a constituent product of T cells.

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.

Production of interleukin 2 and interleukin 4 by immune CD4-CD8+ and their role in the generation of antigen-specific cytotoxic T cells.

In this report we investigated the production and role of interleukin (IL)2 and IL4 in the generation of antigen-specific cytotoxic T cells (CTL). We used as our model the ultraviolet-light-induced epithelial tumor 1591, a highly immunogenic regressor tumor which evokes a strong cell-mediated immune response leading to rejection. We show that IL2 and IL4 are differentially required for the development of optimal cytolytic activity to the 1591 tumor in primary and secondary in vitro splenic cultures. First, anti-IL2 receptor monoclonal antibody (mAb) significantly decreased specific cytotoxicity in both primary and secondary splenic mixed lymphocyte-tumor cell culture (MLTC) cultures, but anti-IL4 mAb inhibited the cytotoxic responses only secondary and not primary cultures. Second, when supernatants from MLTC were tested for lymphokine activity, primary cultures produced only IL2 while secondary cultures produced both IL2 and IL4. Splenic cells were then depleted of CD4+ cells by negative selection, or enriched for CD8+ cells by positive selection, and tested for lymphokine production and requirements. CD8+ cells could not generate significant CTL activity in primary cultures, but could in secondary MLTC. The addition of mAb to either IL2 or IL4 significantly inhibited the generation of CTL by CD8+ cells in these secondary MLTC.CD8+ cells were also found to produce both IL2 and IL4 in secondary MLTC by functional and Northern blot analysis. The production of IL2 and IL4 by CD8+ cells occurs during different phases of culture, with IL2 being produced early (days 1 and 2) and IL4 late (days 3-5). In addition, the requirement of CD8+ cells for both IL2 and IL4 is unique for that lymphokine. These results suggest that both IL2 and IL4 are both produced and required by CD8+ cells during secondary MLTC, and suggest an additional cellular source of IL4 production besides CD4+ T cells during antigen-specific CTL responses.

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.

Generation of a T-cell hybridoma producing a contrasuppressor factor for contact sensitivity.

C3H/HeN mice were immunized to induce contrasuppressor T-cell (Tcs) activity, splenic T cells from these mice were fused with the BW5147 thymoma, and the resulting hybridomas were tested for their ability to produce a contrasuppressor T-cell factor (TcsF). Nine TcsF-producing hybridomas were preliminarily identified by their ability to inhibit the effect of antigen-specific suppressor T-cell factor (TsF) on the adoptive transfer of contact sensitivity. One of these hybrids, AF5.C6, was cloned, the production of a contrasuppressor factor confirmed, and the high-titred TcsF produced by this cloned hybrid characterized. Hybridoma-derived TcsF is antigen-specific and specifically binds its antigen, but does not bear immunoglobulin (Ig) determinants. Thus, hybridoma-derived TcsF is serologically and functionally identical to an antigen-specific contrasuppressor factor for contact sensitivity, whose production from splenocyte cell cultures has previously been described. The generation of a hybridoma secreting a contrasuppressor factor identical to that produced by spleen cells significantly strengthens the hypothesis that the phenomenon of T-cell contrasuppression is mediated by a specific subset of cells whose activity is contrasuppressive. The further advantages of employing T-cell hybridomas for functional, biochemical and molecular genetic analyses of contrasuppression are also discussed.

Inhibition of Ly-6A antigen expression prevents T cell activation.

Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression. Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect of antisense oligonucleotides on stimulation of preactivated D10 cells. Preactivation of D10 cells with Con A + IL-1 renders them receptive to secondary stimulation by other lymphokines. In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with IL-2, IL-4 + IL-1, or PMA + ionomycin. We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.

Prevention of tumor growth and enhancement of cell-mediated immunity by an antigen-specific contrasuppressor factor from a T cell hybridoma.

We have produced a contrasuppressor T cell hybridoma which has positive effects on multiple forms of cell-mediated immunity. First of all, it protects the adoptive transfer of contact sensitivity from suppressor cells and factors. In addition, TcsF modifies the response to normally tolerogenic administrations of hapten, leading mice to develop contact sensitivity and CTL activity instead of tolerance. Most relevant to this conference, mice which have been both treated with AF5.C6 TcsF and painted with TNCB resist challenge with highly malignant TNP-modified tumors. These experiments suggest a decisive role for contrasuppression in tumor rejection.

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.