Staphylococcal enterotoxin B and tumor-necrosis factor-α-induced relapses of experimental allergic encephalomyelitis: Protection by transforming growth factor-β and interleukin-10

A study was made of the ability of the superantigen staphylococcal enterotoxin B (SEB) to induce relapses of experimental allergic encephalomyelitis (EAE) in SJL mice that had partially or completely recovered from acute EAE. We find that a single injection of 0.05 mg SEB i.v. induces mild relapses in 50% of such mice. In addition, tumor necrosis factor (TNF)-α (0.2 μg, i.p.) also induces EAE relapses in 43% of SJL mice when injected 1–2 months after recovery. SEB does not induce a second relapse if reinjected when Vβ17a⁺ T cells are still partially deleted. In these mice, however, TNF-α is equally effective in inducing relapses as in mice that did not receive SEB previously. We showed earlier that transforming growth factor (TGF)-β and TNF-α have antagonistic effects on experimental autoimmune diseases; e.g., in spontaneously relapsing EAE, TGF-β and anti-TNF were protective, while anti-TGF-β caused disease exacerbation. Interleukin (IL)-10 is also known to counteract certain TNF effects. We now find that both human IL-10 and TGF-β2 lower the incidence of EAE relapses when given simultaneously with SEB or TNF-α. The protective effect of TGF-β is significant only against relapses induced by SEB (reduced to 9%), and that of IL-10 only against relapses induced by TNF (reduced to 0%) with the treatment regimens employed. Neutralizing anti-TGF-β does not increase the incidence of SEB-induced EAE relapses. In contrast, anti-IL-10 increases both the incidence and the severity of such relapses. We conclude that TNF production is probably important in causing EAE relapses, but that other aspects of the SEB-induced reactivation of myelin-specific T cells also contribute. Furthermore, endogenous IL-10 rather than TGF-β production appears to limit the susceptibility to induction of EAE relapses in this model.     

Interleukin-1β, interleukin-6, epidermal growth factor and transforming growth factor-α are elevated in the brain from parkinsonian patients

Interleukin (IL)-1β, IL-6, epidermal growth factor (EGF), and transforming growth factor-α (TGF-α) were measured for the first time in the brain (caudate nucleus, putamen and cerebral cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme immunoassays. The concentrations of IL-1β, IL-6, EGF, and TGF-α in the dopaminergic, striatal regions were significantly higher in parkinsonian patients than those in controls, whereas those in the cerebral cortex did not show significant differences between parkinsonian and control subjects. Since these cytokines and growth factors may play important roles as neurotrophic factors in the brain, the present results suggest that they may be produced as compensatory responses in the nigrostriatal dopaminergic regions in Parkinson's disease, and may be related, at least in part, to the process of neurodegeneration in Parkinson's disease.     

Role of transforming growth factor-βthe preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B

Stimulation of murine CD4⁺ T cells with staphylococcal enterotoxin B (SEB) results in the preferential development of T helper (Th) 1 cells [i.e. high interferon (IFN)-γ and low interleukin (IL)-4, IL-5 and IL-10]; whereas in response to plate-bound anti-CD3 or anti-T cell receptor-αβ, Th1 as well as Th2 cells develop. In the present study, we examined the mechanism which is responsible for the selective Th1 development in the SEB system. The addition of IL-4 resulted in a strong development of Th2 cells showing that SEB stimulation can result inTh2 differentiation. Co-stimulation with anti-CD28 was insufficient in this regard. Lack of Th2 development in the SEB system was in part due to the inhibitory effect of endogenously produced transforming growth factor-β (TGF-β), because anti-TGF-β allowed the development of Th2 cells. Similarly, TGF-β inhibited Th2 development and stimulated Th1 development in the anti-CD3 system. This shift was only partially prevented by also including IL-4 in the cultures. The effects of TGF-β could only partially be explained by stimulation of IFN-γ or inhibition of IL-4 as intermediatory cytokines: (1) TGF-β stimulated Th1 development even in the presence of anti-IL-4 and anti-IFN-γ, and (2) a strong inhibitory effect of anti-TGF-β on Th1 development was still observed when anti-IL-4 and IFN-γ were simultaneously added to the cultures. It is concluded that SEB favors Th1 development by stimulation of TGF-β production. Inhibition of Th2 development by TGF-β is due, in part, to inhibition of IL-4 and stimulation of IFN-γ, and, in part, to a direct effect of TGF-β on the responding T cells.     

Induction of macrophage nitric oxide production by interferon-γ and tumor necrosis factor-α is enhanced by interleukin-10

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-γ (IFN-γ)-stimulated macrophages (MΦ) by preventing the secretion of tumor necrosis factor-α (TNF-α) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-γ together with exogenous TNF-α to induce NO synthesis by bone marrow-derived MΦ. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO production by MΦ stimulated in the presence of optimal concentrations of prostaglandin E₂, a positive modulator of MΦ activation by IFN-γ/TNF-α. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-γ/TNF-α cultures and enhanced NO release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on MΦ function are more complex than previously recognized.     

CD32 expression and signaling is down-regulated by transforming growth factor-β1 on human monocytes

CD32 (FcγRII) is the most abundantly distributed class of IgG Fc receptors in the human body. In this study, we analyzed the effect of transforming growth factor (TGF)-β1, a cytokine with strong immunosuppressive function, on the expression and function of CD32 on freshly isolated peripheral blood monocytes and three human monocytic cell lines, U937, THP-1 and Mono mac-6. We found that TGF-β1 down-regulates CD32 expression on monocytes and all monocytic cell lines in a dose- and time-dependent fashion. A mean down-regulation of CD32 expression on THP-1 cells of 54 ± 3.2% after 24 h was found at a concentration of 1 ng/ml TGF-β1. At the mRNA level, TGF-β1 induced a twofold down-regulation of CD32. Cross-linking of CD32 induced an increase in the concentration of intracellular Ca²⁺, which was reduced by 50% by TGF-β1, suggesting a decreased downstream signaling mediated by the receptor.     

IFN-γ inhibits the production of latent transforming growth factor-β1 by mouse inflammatory macrophages

Transforming growth factor (TGF)-β is a multifunctional cytokine, which in mammals exists in three isoforms (TGF-β1, 2 and 3). It is synthesized by a variety of cells including macrophages, and exerts potent immunoregulatory effects such as the inhibition of Th1 development and the suppression or reversal of IFN-γ-induced macrophage activation. In this study we analyzed the effect of IFN-γ on the production of TGF-β1 by thioglycolate-elicited mouse peritoneal macrophages under serum-free conditions. Untreated macrophages released TGF-β1 in its latent form, which became detectable in a capture ELISA specific for active TGF-β1 after acid activation of the culture supernatants. Treatment with IFN-γ reduced the amount of latent TGF-β1 in the culture supernatants in a dose-dependent fashion. The effect of IFN-γ was confirmed by a newly developed Western blot system for the detection of mouse TGF-β1 protein. IFN-γ only weakly (16 – 24 %) reduced the levels TGF-β1 mRNA at early and late time points of stimulation, and no evidence was obtained that IFN-γ suppresses the secretion of latent TGF-β1. Thus, inhibition of TGF-β1 production by IFN-γ is most likely due to decreased synthesis and/or stability of the TGF-β1 protein, and might be important for the generation of fully activated macrophages and a Th1 response.     

Differential effects of interleukin-12 on the development of naive mouse CD4+ T cells

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4⁺ T cells was studied in an accessory cell-free in vitro system. Dense CD4⁺ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-γ after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-γ and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4⁺ T cells support the development of T_H1 and T_H2 cells, respectively. When naive CD4⁺ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on T_H1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced T_H2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-γ by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-γ production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of T_H1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of T_H2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of T_H0 cells.     

Interferon-γ-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-γ production

The novel cytokine interferon-γ-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-γ (IFN-γ) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-γ had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-γ; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-γ production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-γ, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-γ production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.     

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-α and interferon-γ

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), we investigated the effects of these two cytokines on death of luteal cells in vitro. Methods: Mouse luteal cells were cultured in serum-free medium with TNF-α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN-γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. Results:On day 3 of culture, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-γ (1,000 U/ml) and TNF-α (5,000 U/ml) did. On day 6, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-α alone at 5,000 U/ml did, and combinations of IFN-γ and TNF-α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-α. On days 3–6 of culture, combinations of IFN-γ and TNF-α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. Conclusions: The presence of IFN-γ modulates the ability of TNF-α to induce a reduction in the number of viable cells, although TNF-α alone at high concentrations can induce a reduction in the number of viable cells. © 1995 Wiley-Liss, Inc.     

Interleukin-12 activates human γδ T cells: synergistic effect of tumor necrosis factor-α

γδ T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood γδ T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for γδ T cell activation in vitro: interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated γδ T cells, but not αβ T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed γδ T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-α monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on γδ T cells, suggesting that endogenous TNF-α may play a role in IL-12-induced activation of γδ T cells. Recombinant TNF-α synergistically augmented IL-12-induced activation of γδ T cells. Furthermore, IL-12 up-regulated TNF receptors on γδ T cells in vitro: TNF-α binding to its receptor induced CD25 expression on the γδ T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-α were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, γδ T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-α, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.     

Transforming growth factor-β1-induced expression of the mucosa-related integrin αE on lymphocytes is not associated with mucosa-specific homing

The integrin α_E (HML-1, α_IEL, α_M290) is largely expressed on lymphocytes in epithelial sites, especially the gut mucosa. We investigated whether α_E has any role in homing or delineates a phenotype with distinct migratory behavior. Lymph node T cells were stimulated for 5 days with anti-CD3 in the presence or absence of transforming growth factor (TGF)-β₁ to generate α_E⁺ or α_E^? cells, respectively. The two populations were then tested for their homing properties in mice. Both α_E⁺ (TGF-β-treated) and α_E^? (control) cells of either CD4⁺ or CD8⁺ subset had a low capacity to enter the gut and showed the same homing behavior with respect to a variety of other organs. The same was true for α_E⁺ and α_E^? cells that had been briefly stimulated with anti-CD3 (24 h) and then allowed to return to a resting state before injection, though in this case both populations showed a greater capacity to recirculate through lymphoid tissue than was seen with fully activated cells. The results indicate that α_Eβ₇ does not act as a homing receptor, and that the expression of the site-specific marker α_E does not correlate with a distinct homing behavior.     

Collagen synthesis of human arterial smooth muscle cells: Effects of platelet-derived growth factor,transforming growth factor-β1 and interleukin-1

The effects of platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1) and interleukin-1 (IL-1) on collagen synthesis of cultured human arterial smooth muscle cells in a confluent state were investigated. Synthetic activity of collagenous protein was determined with [³H]-proline uptake, and subsequent analysis of collagen types by sodium dodecylsulfte-polyacrylmide gel electrophoresis (SDS-PAGE) followed by fluorography. Although PDGF (0.5 U/mL and 5.0 U/mL) enhanced total collagen synthesis per dish, it suppressed total collagen synthesis per DNA (DNA content in a dish). TGF-β1 (10 pmol/L and 100 pmol/L) enhanced total collagen synthesis both per dish and per DNA. IL-1 (0.1 U/mL and 1.0 U/mL) suppressed total collagen synthesis both per dish and per DNA. A fluorogram revealed that human arterial smooth muscle cells synthesize types I, III, IV and V collagen. Densitometric analysis showed PDGF suppressed the proportion of type V collagen. TGF-β1 increased the proportions of types IV and V collagen. IL-1 elicited un-remarkable change in the proportion of collagen types. These results suggest that, in the event of human atherosclerosis, TGS-β1 is most effective in enhancing collagen synthesis, and PDGF modulates collagen metabolism by stimulating a cell division of smooth muscle cells with a resultant increase of collagenous protein, especially of type V collagen.