T cell anergy is programmed early after exposure to bacterial superantigen in vivo

We have investigated the effects of the protein synthesis inhibitor,cycloheximide (CHX), on the induction of post-thymic T celltolerance in mice primed with the bacterial superantigen, Staphylococcusaureus enterotoxin B (SEB). A single injection of 1 mg CHX preventedprotein synthesis in splenic cells for <6 h in vivo. Theconcomitant administration of SEB and CHX prevented inductionof SEB-specific anergy, but did not interfere with the deletionof SEB-speclfic V_ß8⁺ T cells by activation-induced,programmed cell death. When CHX was given 24 h after SEB administrationthe expression of anergy was not affected. These findings suggestthat anergy and deletion represent independent processes. Furthermore,these observations, together with the fact that SEB retainsthe potential to induce anergy in specific T cells 8 h afterpriming in vivo, imply that the determination of alternate fates(anergy or death) occurs at early time points after SEB injection     

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V_β8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V_β8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing “anergy” and apoptotic cell death of V_β8⁺ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V_β8⁺ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)α chains in vivo, although SEB induces IL-2R α in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V_β8⁺ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.     

Role of nitric oxide in activation of human T lymphocytes induced by bacterial superantigen

The involvement of nitric oxide (NO) in the regulation of human T cell response to bacterial superantigen (staphylococcal enterotoxin B) was studied. It was shown that stimulated T lymphocytes are the main source of NO. This superantigen markedly increased NO production and triggered the proliferative response of mononuclear cells from healthy individuals; the degree of apoptosis was low. In patients with purulent surgical diseases with high spontaneous and induced NO production, superantigen enhanced apoptosis of lymphocytes and induced anergy of T cells to enterotoxins. Increasing the concentration of NO in cultured cells from healthy individuals in the presence of NO donors also stimulated apoptosis and inhibited proliferative activity. These data suggest that NO regulates T lymphocyte response to superantigens. The increased production of NO probably contributes to the development of immunosuppression during bacterial infection. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 10, pp. 402–406, September, 2000     

Mycobacterium avium infection of macrophages results in progressive suppression of interleukin-12 production in vitro and in vivo.

Interleukin-12 (IL-12) has been shown to have an important role in the host defense against Mycobacterium avium. We sought to determine if human monocyte-derived macrophages produce IL-12 upon M. avium infection. Although IL-12 can be measured in supernatants of M. avium-infected macrophages at 24, 48, and 72 h following infection, intracellular staining showed that 24 to 48 h after infection, IL-12 was synthesized chiefly by uninfected macrophages in the monolayer, suggesting that M. avium infection inhibits IL-12 production. In addition, the data also suggest that the longer macrophage monolayers were infected, the less IL-12 they were able to produce. Stimulation of macrophages with IFN-gamma prior to infection with M. avium resulted in greater production of IL-12 compared with unstimulated macrophages. Culture supernatant of M. avium-infected macrophage monolayers, but not control macrophages, partially inhibited IL-12 production by IFN-gamma-stimulated macrophages. This partial inhibition was not reversed by anti-interleukin-10 (anti-IL-10) and anti-transforming growth factor beta 1 (anti-TGF beta 1)-neutralizing antibodies. M. avium infection of macrophages in vitro also suppressed IL-12 synthesis induced by Listeria monocytogenes infection. Immunohistochemistry staining of spleen of infected mice showed that IL-12 production by splenic macrophages was more pronounced in the beginning of the infection but decreased later. Our data indicate that M. avium infection of macrophages suppresses IL-12 production by infected cells and that the suppression was not a result of the presence of IL-10 and TGF beta 1 in the culture supernatant.     

Evidence that ultraviolet-light induced DNA replication death of rec A bacterial is prevented by protein synthesis in repair-proficient bacteria

The utlraviolet light (UV) survival curve of Escherichia coli WP10 recA trp is almost biphasic, with a greatly reduced shoulder but demonstrating a transition to a decreased slope with increasing fluences, indicating the presenc ein the culture of a low freqeuency of resistant cells. Treatment of the culture with chloramphenicol before UV exposure brought almost all the cells to a high degree of UV resistance, by bringing them to the end of their DNA replication cycle. The survival curves of the repair-proficient E. col WP2 trp showed a similar pattern with chloramphenicol treatment of tryptophan starvation before UV exposure, but only if protein synthesis were blocked by chloramphenicol for 60 min after UV exposure. The result suggests that when recA/lexA-regulon induction is prevented, either by the recA mutation or by inhibition of protein synthesis after UV exposure, death occurs unless the cells are in the resitant state characteristic of bacteria at the end of their DNA replication cycle. With repair-proficient bacteria treated before UV exposure with chloramphenicol, when protein synthesis is not blocked after UV exposure, a marked expansion of the shoulder occures because of the function of another resistance-conferring mechanism. This mechanism also depends on the recA⁺ gene expansion of the shoulder does not occur in recA bacteria when protein synthesis is inhibited before UV exposure.     

Clonal deletion as direct consequence of an in vivo T cell response to bacterial superantigen

To date clonal deletion of peripheral mature T cells is restricted to in vivo model systems characterized by prolonged exposure of mice to antigens and clonal T cell expansion preceding clonal deletion. Here we describe that upon challenge of mice with the superantigen staphylococcal enterotoxin B two immediate events become imposed on ligand-reactive Vβ8⁺ T cells in lymph node cells draining the local site of injection. First, and within hours Vβ selective clonal deletion is initiated via an apoptotic process. Second, the remaining Vβ8⁺ T cells first develop a profound state of ligand-specific unresponsiveness and subsequently initiate clonal in vivo growth. It is suggested that the dichotomy of events observed reflects a direct consequence of T cell receptor occupancy in the context of inappropriate signalling.     

Enhanced resistance of mice to bacterial infection induced by recombinant human interleukin-1a.

The effect of recombinant human interleukin-1a on the survival rate of Std-ddY male mice systemically infected with Pseudomonas aeruginosa 12 or Klebsiella pneumoniae P-5709 was evaluated. In P. aeruginosa infection, interleukin-1a given intramuscularly twice, 3 days and 1 day before inoculation of bacteria, most effectively protected animals from death due to infection. The effect was dose dependent, with a maximum survival rate of 92.5% at 10 micrograms per mouse, while only 8.3% of the control group survived until the end of the observation period. The 50% effective dose of interleukin-1a was 0.261 microgram per mouse. In K. pneumoniae infection, interleukin-1a given intramuscularly twice, simultaneously with and 1 day after the inoculation of bacteria, was most effective. The protective effect of interleukin-1a was again dose dependent and was generally more marked than in P. aeruginosa infection. The 50% effective dose was 0.034 microgram per mouse. In both infections, there was no significant increase in the survival rates of animals injected with human albumin or heat-inactivated interleukin-1a. These observations raise the possibility that human interleukin-1a could serve as a therapeutic tool for patients with bacterial infections.     

Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo.

Interleukin-8 (IL-8) is a neutrophil chemoattractant cytokine. Initially IL-8 appeared to exhibit specificity for neutrophils over other cells of the immune system. However, several recent studies have shown that this mediator can also activate other leucocyte types in vitro. In this study we have used an in vivo model of local [111In]leucocyte accumulation in the guinea-pig and an in vitro assay of leucocyte activation (changes in cytosolic-free Ca2+) to investigate the eosinophil chemoattractant activity of IL-8. The intradermal injection of recombinant human (rh)IL-8 induced a dose-dependent accumulation of intravenously administered [111In]eosinophils into the skin sites over 4 hr. Time-course experiments revealed that this cell infiltration was delayed in onset, occurring between 1 and 2 hr after injection of IL-8. The delay may indicate that IL-8 operates via an indirect mechanism. In contrast, eosinophil accumulation induced by the complement fragment C5a occurred within the first hour following injection. Other human cytokines, IL-1, IL-3, IL-5, tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were not eosinophil chemoattractants in this in vivo test system. Direct activation of eosinophils by IL-8 was demonstrated in vitro by a transient elevation in cytoplasmic-free Ca2+ levels where it was less potent than either rhC5a or leukotriene B4 (LTB4). Experiments using [111In]neutrophils in vivo indicated that rhIL-8 and rhC5a were similar in potency in inducing local neutrophil infiltration into guinea-pig skin. The demonstration of the eosinophil chemoattractant activity of IL-8 in vivo raises the possibility that this cytokine, or a structurally related molecule, contributes towards eosinophil infiltration in a number of inflammatory conditions such as asthma, helminthic infections and adult respiratory distress syndrome.     

Endometrial ability to implant in ectopic sites can be prevented by interleukin-12 in a murine model of endometriosis

Immune dysfunctions in endometriosis are widely documented but the effectiveness of immunotherapies for the management of the disease is still debated. Progress in this field has also been limited by the lack of an appropriate animal model of the disease. In this study, we created a model of endometriosis in immunocompetent mice to verify the ability of endometrium to implant in ectopic sites and to investigate the potential application of the cytokine interleukin (IL)-12 in preventing this ectopic implantation. Endometriotic lesions were induced in both C57BL/6 and BALB/c mice by inoculating syngenic endometrial fragments through a small laparotomic incision into the peritoneal space. All the animals challenged with syngenic endometrium showed evidence of peritoneal endometriosis at 3 weeks. Histologically, endometriotic lesions consisted of cystic endometrial glands surrounded by a stroma. Intraperitoneal injection of IL-12 was able to reduce total weight and total surface area of endometriotic lesions respectively of 77 and 61% in C57BL/6 and of 42 and 28% in BALB/c mice. These results demonstrate that IL-12 is able to induce a significant prevention of ectopic endometrial implantation in an in-vivo model of endometriosis. These findings support the possibility of using the immune system to generate novel therapies for the management of the disease.     

Endogenous interleukin-12 is involved in resistance to Brucella abortus infection.

Protective immunity against Brucella abortus is mediated by acquired cellular resistance, with gamma interferon (IFN-gamma)-producing T cells playing a key role. Interleukin-12 (IL-12) is a cytokine that has a profound effect on the induction of IFN-gamma-producing Th1 and NK cells. Here we report that depletion of endogenous IL-12 before infection of mice significantly exacerbated brucella infection. IL-12-depleted mice also had reduced splenomegaly resulting from infection and showed a decrease in percentage and absolute numbers of macrophages compared with those in control infected mice. Furthermore, spleen cells from IL-12-depleted mice had a reduced ability to produce nitrite, a product of activated macrophages. This could be the result of the low production of IFN-gamma by splenic T cells observed in the IL-12-depleted mice. The mechanism whereby IL-12 controls antibacterial resistance is discussed.     

Cytogenetic damage induced in vivo to mice by single exposure to cesium chloride

Female laboratory bred albino mice (2n = 40) were orally administered cesium chloride (CsCl) in aqueous solution as a single dose and the damage induced at the chromosomal level was observed in bone marrow cells after 6, 12, 18, and 24 hours of exposure. The concentrations of the chemical given were calculated as fractions of the LD50, namely 1/5, 1/10, and 1/20. The cytogenetic endpoints screened for were chromosomal aberrations (CA) and divisional frequency or mitotic index (MI). The frequency of chromosomal aberrations induced was directly proportional to the concentration of the chemical administered. The highest dose was the most toxic and was considered to be the maximum tolerance level. Effects on divisional frequency were variable, the highest concentration being significantly mitostatic, the middle one ineffective, and the lowest slightly mitogenic. In general, the observations indicate that CsCl is clastogenic when administered orally to mice in vivo and the effects are dose-dependent.     

Tolerance to appetite suppression induced by peptidoglycan.

Physiologically realistic peptidoglycan (PG) fragments, derived from Neisseria gonorrhoeae, were shown previously to dose-dependently suppress food consumption and body weight gain in rats following single intraperitoneal injections. The present study, examining the effects of repeated daily injection of PG, provides additional support to our underlying hypothesis, i.e., that soluble PG fragments contribute to the loss of appetite commonly associated with bacterial infections. An initial intraperitoneal injection of purified, soluble, macromolecular, extensively O-acetylated PG fragments (S-O-PG) (240 micrograms/kg of body weight) decreased overnight food consumption in male Lewis rats (150 g) by approximately 35% relative to animals receiving diluent alone (P < 0.05). However, subsequent daily injections of S-O-PG resulted in progressively smaller effects on food consumption until, by the fourth day, rats were completely nonresponsive (tolerant) to S-O-PG-induced hypophagia. Rats that developed tolerance to the effects of S-O-PG on appetite were also tolerant to three other known hypophagic agents, lipopolysaccharide (LPS), muramyl dipeptide, and interleukin-1, when challenged one day after establishment of S-O-PG tolerance. Similarly, rats developed tolerance to repeated injections of muramyl dipeptide or LPS and were cross-tolerant to S-O-PG when challenged 1 day later. However, 30 days after establishment of S-O-PG tolerance, rats remained nonresponsive to S-O-PG but regained full responsiveness to LPS-mediated hypophagia. Thus, at least two mechanisms of tolerance to S-O-PG hypophagia exist: an early tolerance which is nonspecific and a late tolerance which is specific for S-O-PG. Late, but not early, tolerance to S-O-PG-mediated suppression of appetite was associated with an increase in specific anti-PG antibody activity as measured in an enzyme-linked immunosorbent assay.     

Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12

There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD). In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions. In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ). We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e. are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e. are immune when exposed to bacterial sonicates derived from the heterologous intestine of syngenic littermates (BsH). Furthermore, we demonstrate that both local and systemic tolerance to BsA is broken in a murine model of chronic intestinal inflammation induced by the hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS), which mimics several important characteristics of Crohn's disease. Tolerance to BsA was restored and TNBS-induced colitis was abrogated in mice systemically treated with interleukin (IL)-10 or antibodies to IL-12. Treatment specifically restored tolerance to BsA, but did not suppress proliferation to BsH. In summary, we here report a new mouse model for the study of immunity and tolerance towards bacterial products. Our data suggest that tolerance to BsA is an important protective mechanism and that restoration of tolerance to resident intestinal flora by IL-10 and antibodies to IL-12 may be of potential therapeutic utility in patients with inflammatory bowel disease.     

Characteristics of murine non-specific killer cells induced in vivo by recombinant human interleukin-2.

We have examined the induction of murine non-specific killer cells in vivo and in vitro by purified recombinant human interleukin-2 (rIL-2), and compared their characteristics with respect to killing ability, cell surface phenotypes, and antibody-dependent cell-mediated cytotoxicity (ADCC). C57BL/6 spleen cells cultured with rIL-2 were remarkably cytotoxic against a variety of tumour cells in a 4-hr 51Cr-release assay. Treatment with various antibodies (anti-Thy 1, anti-Lyt 1, anti-Lyt 2, and anti-asialo GM1) plus complement (C) showed that anti-Thy 1 or anti-asialo GM1 antibody plus C removed a majority of killer activity (80% and 66%, respectively). In addition, an increase in ADCC was detected in the spleen cells cultured with rIL-2. These ADCC effector cells were indistinguishable from non-specific killer cells by the cell surface phenotypes. A single administration of rIL-2 in vivo induced only transient and marginal enhancement of non-specific killer activity of spleen cells in C57BL/6 mice. On the other hand, when 10 micrograms of rIL-2 were administered daily by bolus to C57BL/6 mice, the activity increased gradually for about 10 days and reached a plateau. This enhanced non-specific killer activity rapidly decreased and returned to normal by 72 hr after the administration was stopped. The non-specific killer cells induced in vivo in this manner were not only greatly cytotoxic against natural killer (NK)-sensitive tumour cells but were also significantly cytotoxic against NK-resistant tumour cells. Most of the killer activity (more than 90%) was specifically removed by treatment with anti-Thy 1 or anti-asialo GM1 antibody plus C. An increase in ADCC was detected concurrently with an increase in non-specific killer activity in vivo, and both effector cells were indistinguishable by their cell surface phenotypes. These results indicate that a majority of non-specific killer cells induced both in vivo and in vitro by rIL-2 have some common features. Our results also suggest that these cells belong to the same lineage as NK cells, although they are thought to be at different stages from resident NK cells.     

Immune response to intragraft antigen in draining lymph nodes after corneal transplantation is mediated by interleukin-12.

To examine the molecular basis of immunity generated to intragraft antigens and determine whether it differs between acceptor and rejector hosts, we used a novel in vitro system to assay the T cell response to a specific antigen, ovalbumin (OVA), in the graft. OVA-containing corneas were orthotopically grafted into syngeneic or allogeneic hosts. Draining cervical lymph nodes (cLN) were assayed for OVA-specific T cell proliferation and cytokine production. In addition, cytokine production was assayed in cultures of antigen-presented cells (APC) isolated from cLN cultured with OVA-specific DO11.10 T cells and OVA. OVA-specific immunity was detected only in the draining cLN of mice following allogeneic, but not syngeneic, grafting, and this immunity was evident well before any demonstrable alloresponse in the graft. In addition, cLN cells from mice that accepted their corneal allografts produced significantly less interferon-gamma (IFN-gamma) when stimulated in culture than cells harvested from cLN of rejector hosts. Moreover, APC isolated from cLN of acceptor hosts produced significantly lower levels of IL-12. These data suggest that the induction of immunity to corneal antigens in the draining cLN occurs via an interleukin-12 (IL-12) and IFN-gamma-dependent mechanism. Targeting this process may serve as an effective immunomodulatory strategy in corneal transplantation.     

Mechanism of cellular suppression induced by oral tilorone treatment of mice.

Specific-pathogen-free B6D2 F1 hybrid mice were treated orally with tilorone hydrochloride (100 mg/kg of body weight per day) and infected with sublethal doses of Listeria monocytogenes, Salmonella enteritidis, Mycobacterium bovis (BCG Pasteur), or M. tuberculosis Erdman. Daily tilorone treatment inhibited the cell-mediated response to all of the intracellular parasites, and most of the mice succumbed to the challenge. Tilorone suppressed delayed hypersensitivity responses to the microbial sensitins as well as to sheep erythrocytes. However, humoral responses (immediate hypersensitivity reactions) were stimulated. The types of growth curves obtained in the tilorone-treated mice were quite different from those observed in T-cell-depleted mice and tended to resemble those seen in sublethally irradiated (400 rads) animals. Leukocyte counts were depressed 10-fold by daily tilorone treatment. Both monocyte and granulocyte (but not large-lymphocyte) counts were depressed. There was an initial drop in small-lymphocyte counts with a later recovery phase. Tilorone treatment reduced the granulomatous response within the Salmonella-infected liver, suggesting that the drug interferes with the mobilization of the mononuclear defenses within the normal host.     

Increased sensitivity to Salmonella enterica serovar Typhimurium infection in mice undergoing withdrawal from morphine is associated with suppression of interleukin-12

We have shown previously that withdrawal from morphine induces immunosuppression in mice. The present study reports the effects of morphine withdrawal on infection with Salmonella enterica serovar Typhimurium. Mice were made dependent on morphine by the implantation of a slow-release morphine pellet for 96 h. Controls received a placebo pellet. Withdrawal was induced by pellet removal. Mice were inoculated intraperitoneally with Salmonella 24 h postwithdrawal. Morphine withdrawal sensitized mice to Salmonella infection, as evidenced by increased mortality, shortened mean survival time, and increased bacterial load in the blood, spleen, and liver. Examination of the levels of a panel of proinflammatory cytokines in sera of infected, morphine-withdrawn mice showed that morphine withdrawal inhibited the elevation of interleukin-12p70 (IL-12p70). The production of IL-12p40 in morphine withdrawal mice was also suppressed. The administration of exogenous IL-12 significantly decreased the bacterial burden in morphine-withdrawn mice. These studies show a correlation between the suppression of IL-12 production and a heightened susceptibility to Salmonella infection in mice undergoing withdrawal from morphine.     

Alterations in epidermal handling of HSV-1 antigens in vitro induced by in vivo exposure to UV-B light.

In order to investigate the cellular interactions involved in the immune response to herpes simplex virus type 1 (HSV-1) in a murine model, an in vitro antibody induction system was developed. This comprised HSV-1-primed T cells from infected mice, trinitrophenol (TNP)-primed B cells from mice primed with TNP-coupled calf erythrocytes, and TNP-HSV-1 as antigen. When antigen-presenting cells (APC) were removed from the assay system, the induced antibody response disappeared but could be reconstituted by the addition of APC derived from the peritoneal cavity or skin of normal mice. Since HSV-1 is an epidermal pathogen, it was decided to investigate the role of skin APC in HSV-1 immunity. Skin APC from mice irradiated 3 days previously with a suberythemal dose of ultraviolet (UV)-B were found to have a decreased capacity to present HSV-1 antigen in vitro. However, the APC capacity of their peritoneal cells was unaffected. The reduction in APC capacity is not only a local effect at the irradiated site, as APC from mice exposed to UV-B with one ear protected by black electrical tape were equally affected in both ears.