Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kappa B activation.

The glucocorticoid dexamethasone inhibited the production of the rat cytokine-induced neutrophil chemoattractant CINC/gro, a counterpart of human melanoma growth-stimulating activity that belongs to the interleukin-8 (IL-8) family, in the normal rat kidney epithelial cell line NRK-52E stimulated with interleukin-1 beta (IL-1 beta), lipopolysaccharide, or tumor necrosis factor alpha. The accumulation of CINC/gro mRNA induced by these activators was also decreased comparably by dexamethasone. A nuclear run-on assay revealed that dexamethasone decreased the IL-1 beta-induced transcription of the CINC/gro gene. The half-life of CINC/gro mRNA transcripts did not change significantly after exposure to dexamethasone, suggesting that this glucocorticoid acts mainly at the transcriptional level. Transfection with luciferase expression vectors containing 5'-deleted and mutated CINC/gro gene sequences demonstrated that the 5'-flanking region containing the NF-kappa B binding site is involved in the IL-1 beta- and dexamethasone-induced activation and repression of the CINC/gro gene expression, respectively. Furthermore, a tandem repeat of the NF-kappa B sequence in the CINC/gro gene conferred the inducibility by IL-1 beta and suppression of luciferase activity by dexamethasone. In an electrophoretic mobility shift assay, dexamethasone diminished the IL-1 beta-induced formation of NF-kappa B complexes, which consisted of p65 and p50. Western blotting revealed that dexamethasone inhibited the IL-1 beta-induced translocation of p65 from the cytoplasm into the nucleus, while the nuclear level of NF-kappa B p50 remained almost unchanged. In addition, the degradation of I kappa B-alpha induced by IL-1 beta was not inhibited by dexamethasone. These results indicated that the suppression of the CINC/gro gene transcription by glucocorticoid occurs through the impairment of NF-kappa B activation, possibly by interference with the translocation of NF-kappa B p65 from the cytoplasm into the nucleus, thereby suppressing transactivation of the CINC/gro gene.

Level of neutrophil chemotactic factor CINC/gro, a member of the interleukin-8 family, associated with lipopolysaccharide-induced inflammation in rats.

Rat cytokine-induced neutrophil chemoattractant (CINC), which is a counterpart of human gro and belongs to the interleukin-8 family, has been quantified by a new sandwich enzyme-linked immunosorbent assay. Administration of lipopolysaccharide (LPS) into an air pouch performed by subcutaneous injection of air caused inflammation and severe neutrophil infiltration. After the LPS injection, changes in the concentration of CINC/gro, chemotactic activity, and the number of neutrophils in the air pouch exudate were determined. The chemotactic activity of neutrophils was augmented before practical neutrophil infiltration. More than half of the chemotactic activity was neutralized by the antisera. The time kinetics of the level of CINC/gro coincided with the changes in chemotactic activity. The maximal level of rat CINC/gro was 85 ng/ml, which is sufficient to cause neutrophil migration in vitro and in vivo as described previously. These data suggest that rat CINC/gro is a functional chemoattractant for neutrophils in LPS-induced inflammation in rats.

Immunosuppressive activity of serum from liver-grafted rats: in vitro specific inhibition of mixed lymphocyte reactivity by antibodies against class II RT1 alloantigens.

The immunosuppressive activity of serum from PVG rats following orthotopic transplantation of DA liver has been examined in vitro. Liver grafts in this combination are never rejected, but induce a state of specific transplantation tolerance in the recipient. Serum from such tolerant animals was able to inhibit proliferation of normal PVG lymph node cells in response to DA stimulators in the mixed lymphocyte reaction (MLR); inhibition was specific for donor (DA) antigens. Interleukin-2 (IL-2) production during the MLR was also reduced. The production of anti-DA cytotoxic T cells developing in the MLR was not affected, but the total yield of such cells was reduced. Evidence was obtained that part of the inhibitory serum activity was due to IgG antibody against class II RT1a alloantigens. Thus, a purified IgG fraction retained much of the inhibitory activity which could be removed by an anti-IgG absorbent. Studies of MLR inhibition in different rat strains indicated the anti-class II specificity of the inhibitory IgG. Lymph node cells from DA-liver-grafted PVG rats responded normally against DA stimulators in vitro, and this MLR was also blocked by the inhibitory IgG. Our results suggest that anti-class II allo-antibody may play a role in immunosuppression and long-term graft survival following liver transplantation in this combination.

Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells.

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.

Comparison of antigen-specific T cell responses in autoimmune MRL/Mp-lpr/lpr and MRL/Mp-+/+ mice.

The MRL-1 mouse develops severe autoimmune disease characterized by high titers of autoantibodies at an early age (3 to 5 mo). The congeneic MRL-n mouse, which differs only in the lymphoproliferative (lpr) gene, exhibits no such pathologic or serologic abnormalities at the same age. We examined antigen-specific T cell responses in the MRL-1 mouse and compared them to age- and sex-matched MRL-n controls. We found broad defects in these responses in the MRL-1 mouse; an inability to generate primary allospecific and hapten-specific cytolytic T lymphocytes (CTL), secondary hapten- and virus-specific CTL, as well as a deficient proliferative response to hapten and natural antigens and a weak delayed-type hypersensitivity response were demonstrated. Our data furthermore suggest a lack of interleukin 2 (IL 2) acceptor sites in the proliferating T cell, while suggesting no such lack on CTL precursors. In fact, the deficient CTL responses in MRL-1 mice can be restored to levels seen in MRL-n by the in vitro addition of IL 2. The implications of these findings and the possible explanations for the relative deficit in helper function in the MRL-1 mouse are discussed.

Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response.

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.

An antigen-specific signal is required for the activation of second-order suppressor T cells in the regulation of delayed-type hypersensitivity to 2,4,6-trinitrobenzene sulfonic acid.

Suppressor T cells (Ts-1) induced with trinitrophenyl (TNP)-conjugated syngeneic spleen cells (TNP-SC) can be enriched on antigen-coated plates and are afferent suppressors. In addition, these suppressor cells produced soluble suppressor factors (TsF) that were active in vivo. Therefore, the Ts-1 cells in the TNP system are very similar to the Ts-1 cells in other systems we have studied earlier. Further characterization of these TsF-1 revealed that TsF-1 obtained from TNP-SC-induced Ts-1 is major histocompatibility complex restricted in its activity. Injection of TNP-specific TsF-1 into naive mice did not induce Ts-2 unless additional corresponding antigen was provided. Moreover, the Ts-2 cells induced by administration of both TsF-1 and trinitrobenzene sulfonic acid were antigen specific rather than antiidiotypic.

Effect of vesicular stomatitis virus (VSV) infection on the development and regulation of T cell-mediated immune responses.

Infection of mice with vesicular stomatitis virus (VSV) at the time of immunization failed to enhance T cell-mediated immune response to azobenzenearsonate-(ABA) conjugated spleen cells as measured by delayed-type hypersensitivity and by in vitro proliferation and in vitro generation of ABA-specific cytotoxic T cells. However, mice infected with VSV are incapable of responding to signals from suppressor T cells or their soluble factors. Further analysis revealed that VSV infection does not interfere with the induction of Ts-1 or Ts-2 cells. Because infection of Ts-1 or Ts-2 donors had no effect on the subsequent response seen in the recipients of antigen and suppressor T cells, the most likely candidate for the target of VSV infection is therefore the Ts-3 cell or another T cell interacting with Ts-3. This is supported by our observation that it is possible to bypass the VSV effect by providing the recipients of VSV with normal Lyt-2+-bearing T cells.

I-J-restricted interactions in the generation of azobenzenearsonate-specific suppressor T cells.

The genetic restrictions of the activation of third-order suppressor cells (Ts3) were studied in mice, using two different types of anti-azobenzenearsonate (ABA)-immune responses, namely delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) generation. Ts2 cells were induced in several different strains of mice by injecting monoclonal T hybridoma molecules or first-order suppressor factors (TsF1) originating in A/J (H-2a, Igh-1e) mice and then testing the TsF2 molecules derived from these Ts2 in A/J and A.By (H-2b, Igh-1e) or (A/J X A.By)F1 (H-2a/b, Igh-1e) and (C57Bl/6 X A/J)F1 (H-2b/a, Igh-1e) mice. It was shown that the activity of TsF2 was restricted to the I-J of the strain in which Ts2 was induced. By genetic analysis, restriction was shown to be due to the requirement of H-2 identity between ABA-coupled cells used for Ts3 activation and the strain of the TsF2 origin. Moreover, by using H-2-congenic ABA-coupled cells, we were also able to precisely map and demonstrate that ABA-coupled cells I-J identical to TsF2 induced in various strains were necessary for effective suppression to occur. This selective activation of Ts3 suggested the existence of I-J-related antigen presentation for suppression as the counterpart of I-A or I-A-I-E-restricted antigen presentation for positive immune responses.

Two distinct mechanisms regulate the in vivo generation of cytotoxic T cells.

Treatment of responder cells with monoclonal anti-Ly-1,2 antibodies plus complement in vitro completely eliminated their ability to generate azobenzenearsonate (ABA)-specific cytolytic T lymphocytes (CTL). However, addition of the concanavalin A-stimulated supernatants of rat spleen cells (Con A-Sup) can fully reconstitute the response. Therefore, Lyt-1,2-bearing T cells are required for the generation of ABA-specific CTL, and such requirement can be replaced by factors present in the Con A- sup. Suppressor T cells (Ts), when adoptively transferred into naive recipients, will inhibit the in vivo priming of CTL. This inhibition can also be reversed by in vitro addition of Con A-Sup. furthermore, mice serving as donors of Ts also show profound unresponsiveness when primed and restimulated in vitro. In contrast to the Ts-mediated inhibition, in vitro addition of Con A-Sup was unable to abolish the unresponsiveness observed in these cultures. Thus, we identified two unresponsive states in a hapten-specific killing system that differ in their ability to be reconstituted by Con A-Sup.