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M Wysocka

Publications and source records attributed to M Wysocka.

At least 37 records · Page 2Linked to original sources
PubMed1998

Interleukin 12 breaks ultraviolet light induced immunosuppression by affecting CD8+ rather than CD4+ T cells.

Recent studies showed that injection of interleukin (IL)-12 prevents ultraviolet (UV) light mediated suppression of contact hypersensitivity and breaks UV-induced hapten specific tolerance. UV-mediated suppression can be adoptively transferred by injecting splenocytes from UV-irradiated mice; however, suppression is not transferable when donor mice are treated with IL-12 after UV-irradiation. This study was performed to elucidate the mechanisms by which IL-12 counteracts this immunosuppression. To characterize the cells transferring suppression, depletion studies were performed revealing that UV-induced suppression is transferred via CD8+ T cells. To investigate whether IL-12 counteracts UV-induced suppression by either inhibiting the development of CD8+ suppressor T cells or inducing CD4+ effector T cells, splenocytes from mice, which were IL-12 treated and sensitized through UV-exposed skin, were depleted from CD4+ T cells and transferred into naive mice that were subsequently sensitized. Whereas transfer of splenocytes from UV-irradiated mice inhibited sensitization of recipients, no inhibition was observed after transfer of splenocytes from UV-exposed and IL-12 treated mice. Recipients that received CD4 depleted spleen cells from UV-exposed and IL-12 treated donors, were still fully sensitizable. IL-12 also blocked transfer of UV-induced suppression when it was injected into UV-exposed donor animals at a time point when suppressor cells had already developed. CD4 depletion of such splenocytes did not result in a loss of the reconstitutive effect of IL-12. This suggests that IL-12 may break UV-induced tolerance not by inducing CD4+ effector T cells, but rather by inhibiting or inactivating suppressor T cells belonging to the CD8 subtype.

Adoptive Transfer↗
PubMed1997

Inhibition of IL-12 production by thalidomide.

The immunomodulatory properties of thalidomide are currently being exploited therapeutically in conditions as diverse as erythema nodosum leprosum, chronic graft-vs-host disease, rheumatoid arthritis, and sarcoidosis. The relevant mechanism of action of thalidomide in these diseases remains unclear. The important role recently ascribed to IL-12, a cytokine critical to the development of cellular immune responses, in the pathogenesis of several of these conditions led us to examine whether thalidomide affects the production of IL-12. Thalidomide potently suppressed the production of IL-12 from human PBMC and primary human monocytes in a concentration-dependent manner. Thalidomide-induced inhibition of IL-12 production was additive to that induced by suboptimal inhibiting doses of dexamethasone, and occurred by a mechanism independent of known endogenous inhibitors of IL-12 production. These results suggest that thalidomide may have therapeutic utility in a wide range of immunologic disorders that are characterized by inappropriate cellular immune responses.

Antibodies, Monoclonal↗
PubMed1997

The effect of interleukin 12 desensitization on the antitumor efficacy of recombinant interleukin 12.

Use of the cytokine interleukin 12 (IL-12) has been shown to enhance the rejection of a variety of murine tumors, but preclinical and clinical studies have revealed that recombinant IL-12 (rlL-12) can produce severe toxicity. In an effort to improve the tolerance and therapeutic effectiveness of this cytokine, we investigated the influence of giving a single dose of recombinant murine IL-12 (rmIL-12) a week prior to daily cytokine administration (predosing) on its toxic and antitumor effects. These studies were performed in C3H/HeN mice, in which a course of rmIL-12 at standard doses without predosing induced rejection of syngeneic K1735 melanomas in 33%, and in A/J mice, in which treatment induced rejection of syngeneic B7-1+ SCK (SCK.B7-1) mammary carcinomas in 63%. Administration of a predose of rmIL-12 markedly reduced cytokine toxicity in a dose-dependent manner and allowed safe administration of up to 8-fold higher doses of daily rmIL-12 in C3H/HeN mice and 4-fold higher doses of rmIL-12 in A/J mice. Predosing followed by either standard or high daily doses of rmIL-12 did not significantly alter most end points of rmIL-12 treatment of K1735 or SCK.B7-1 tumors (survival, death from tumor, development of protective immunity, and so on), but they appeared to attenuate early control of tumorigenesis by rmIL-12. Evidence for the latter comes from a shortening of the characteristic rmIL-12-induced delay in tumor appearance and in the frequent appearance of tumors that subsequently regress. However, higher doses appear to produce better therapeutic results than standard doses of rmIL-12 after predosing. Predosing severely blunted induction of serum IFN-gamma levels by rmIL-12, which probably accounts for many of the effects of predosing on rmIL-12 toxicity and efficacy. Thus, predosing desensitizes mice to the toxic effects of rIL-12 and allows much higher doses to be given but, despite this, it does not improve and, by some criteria, it attenuates rIL-12 therapeutic outcome. Our results do not support the use of predosing as a way to enhance the effectiveness of rIL-12 in cancer clinical trials.

Animals↗
PubMed1997

B cells regulate CD40 ligand-induced IL-12 production in antigen-presenting cells (APC) during T cell/APC interactions.

Although stimulation of freshly isolated murine spleen cells with anti-CD3 mAb or Con A failed to generate IL-12 production, the same cell preparations depleted of B cells produced IL-12. Addition of normal B cells inhibited IL-12 production in a cell number-dependent manner. IL-12 production was dependent on the presence of CD4+, but not of CD8+, T cells, and inhibited by addition of anti-CD40 ligand (CD40L) mAb. Anti-CD3 or Con A stimulation induced CD40L expression only on CD4+ T cells, which was inhibited in the presence of B cells. IL-12 production was also induced by interactions between CD40L-transfected Chinese hamster ovary cells and splenocytes depleted of T and B cells, but not of APC, indicating CD40L-induced IL-12 production by APC. The involvement of CD40 molecules was examined by comparing the ability of cells from CD40-deficient (CD40 -/-) and wild-type mice (CD40 +/+) to produce IL-12. Spleen cells from CD40 -/- and CD40 +/+ mice produced comparable amounts of IL-12 in response to bacterial stimuli. However, the B cell-depleted fraction from CD40 -/- mice failed to produce IL-12 when stimulated with anti-CD3 or Con A or when cocultured with CD40L-expressing Chinese hamster ovary cells. These results indicate that CD40L expressed on activated T cells induces APC to produce IL-12 through CD40/CD40L interaction, but this pathway is competitively inhibited by CD40+ B cells incapable of producing IL-12 upon stimulation with CD40L. Thus, this might represent a novel mechanism underlying the regulation of cell-mediated and humoral immunity.

Animals↗
PubMed1997

Regulation of T cell-dependent and -independent IL-12 production by the three Th2-type cytokines IL-10, IL-6, and IL-4.

The production of IL-12 by macrophages/dendritic cells (Mphi/DC) is mediated either by a T cell-dependent pathway that is induced primarily by the interaction of CD40 ligand (CD40L) on activated T cells with CD40 on IL-12-producing cells or by a T cell-independent pathway that is induced by bacteria or bacterial products and enhanced by interferon-gamma (IFN-gamma). In this study we investigated the ability of the Th2-type cytokines interleukin (IL)-10, IL-6, and IL-4 to modulate IL-12 production in Mphi/DC induced through the two pathways. IL-12 production was induced in Mphi/DC from normal mice by stimulation with the combination of IFN-gamma plus lipopolysaccharide (LPS) or Staphylococcus aureus Cowan I (a model for the T cell-independent pathway) or by co-culture with Chinese hamster ovary (CHO) cells transfected with the CD40L (a model for the T cell-dependent pathway). The effects of three Th2-type cytokines on IL-12 production by Mphi/DC through the two pathways were examined. IL-10 inhibited IL-12 production induced through both pathways, although the inhibitory effect was more potent on the (IFN-gamma + LPS)-induced pathway. IL-6 inhibited only (LPS + IFN-gamma)-induced IL-12 production. The effect of IL-4 was particularly noteworthy: this cytokine inhibited (LPS + IFN-gamma)-induced IL-12 production, whereas it potentiated the production of IL-12 induced by CD40L. Regulation of IL-12 protein production by IL-10 and IL-4 was found to correspond to the levels of mRNA accumulation for the p40 and p35 IL-12 genes, whereas the presence of IL-6 during stimulation decreased IL-12 protein production without affecting steady-state mRNA levels. These results indicate that IL-12 production in Mphi/DC induced through a T cell-dependent or -independent pathway is positively or negatively regulated by particular cytokines at various control levels.

Animals↗
PubMed1997

Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity.

We previously demonstrated that proliferation of terminally differentiated Th1 clones depends primarily on an interleukin-12 (IL-12)-paracrine mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Th1 clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) alpha beta+, Vbeta11+, NK-1.1-; CD4-CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-gamma (IFN-gamma) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity.

Animals↗
PubMed1997

Inhibition of human interleukin-12 production by pentoxifylline.

Pharmacological control of interleukin-12 (IL-12) production may be a key therapeutic strategy for modulating immunological diseases dominated by type-1 cytokine responses. In this study, we investigated the effects of pentoxifylline on the production of IL-12 by human blood mononuclear cells and primary human monocytes stimulated with heat-killed Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS). Pentoxifylline potently suppressed production of IL-12 in a concentration-dependent manner. In these same experiments, tumour necrosis factor-alpha (TNF-alpha) production was inhibited and IL-10 and prostaglandin E2 (PGE2) production was enhanced by treatment with pentoxifylline. Suppression of IL-12 production by pentoxifylline was found to be independent of several known endogenous inhibitors of IL-12, such as IL-10, transforming growth factor-beta (TGF-beta), IL-4 and PGE2. RNase protection assays revealed that pentoxifylline inhibited accumulation of both IL-12 p40 and p35 mRNA, suggesting a predominant mRNA locus for pentoxifylline-induced IL-12 inhibition. Low levels of pentoxifylline added to the suppression of IL-12 production by suboptimal inhibiting doses of dexamethasone, suggesting that this drug combination may have therapeutic utility. These results provide a firm rationale for the use of pentoxifylline in clinical trials of immunological disorders characterized by inappropriate type-1 immune responses.

Cell Culture Techniques↗
PubMed1996

In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha.

To examine the function of IL-10 synthesis during early infection with the intracellular protozoan Toxoplasma gondii, IL-10 knockout (KO) mice were inoculated with an avirulent parasite strain (ME-49). In contrast to control littermates that displayed 100% survival, the IL-10-deficient animals succumbed within the first 2 wk of the infection, with no evidence of enhanced parasite proliferation. The mortality in the IL-10 KO mice was associated with enhanced liver pathology characterized by increased cellular infiltration and intense necrosis. Levels of IL-12 and IFN-gamma in sera of infected IL-10-deficient animals were four- to sixfold higher than those in sera from control mice, as were mRNA levels for IFN-gamma, IL-1 beta, TNF-alpha, and IL-12 in lung tissue. Similarly, macrophages from IL-10 KO mice activated in vitro or in vivo with T. gondii produced higher levels of TNF-alpha and IL-12 than macrophages from control animals. Moreover, spleen cells from IL-10 KO mice infected with T. gondii secreted more IFN-gamma than splenocytes from nondeficient animals. In vitro depletion experiments indicated that CD4+ lymphocytes are the major source of the latter cytokine in the spleen cell populations, and in vivo depletion with anti-CD4 Abs protected the IL-10 KO mice from parasite-induced mortality. Together the data suggest that endogenous IL-10 synthesis plays an important role in vivo in down-regulating monokine and IFN-gamma responses to acute intracellular infection, thereby preventing host immunopathology.

Acute Disease↗
PubMed1996

Mechanism of suppression of cell-mediated immunity by measles virus.

The mechanisms underlying the profound suppression of cell-mediated immunity (CMI) accompanying measles are unclear. Interleukin-12 (IL-12), derived principally from monocytes and macrophages, is critical for the generation of CMI. Measles virus (MV) infection of primary human monocytes specifically down-regulated IL-12 production. Cross-linking of CD46, a complement regulatory protein that is the cellular receptor for MV, with antibody or with the complement activation product C3b similarly inhibited monocyte IL-12 production, providing a plausible mechanism for MV-induced immunosuppression. CD46 provides a regulatory link between the complement system and cellular immune responses.

Antibodies, Monoclonal↗
PubMed1996

Enhanced expression of IL-12 associated with Th1 cytokine profiles in active pulmonary sarcoidosis.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology characterized by the expansion of activated oligoclonal CD4+ T cells and macrophages at sites of disease. To investigate the immunopathogenesis of sarcoidosis, we analyzed patterns of cytokine expression in bronchoalveolar lavage cells and fluid from patients with pulmonary sarcoidosis and idiopathic pulmonary fibrosis and from normal volunteers. We found dominant type 1 cytokine expression, with elevated mRNA and protein levels of IFN-gamma, but not IL-4, in sarcoid lung cells and fluid compared with those in normal samples. To define immunoregulatory mechanisms important to this type 1 response, we analyzed the expression of IL-12 and IL-10 in lung cells and fluid. Using semiquantitative PCR, we found significantly higher mRNA expression of the regulated IL-12 p40 subunit, but not IL-10, in sarcoid compared with normal lung cells. Consistent with these observations, strikingly elevated levels of p40 protein were found in sarcoid compared with normal bronchoalveolar lavage fluid. Unstimulated and Staphylococcus aureus-stimulated sarcoid alveolar macrophages produced greater amounts of IL-12 than normal alveolar macrophages when cultured in vitro. We hypothesize that sarcoidosis is a Th1-mediated disease driven by chronic, dysregulated production of IL-12 at sites of disease.

Adult↗
PubMed1996

IL-12 produced by antigen-presenting cells induces IL-2-independent proliferation of T helper cell clones.

We investigated the role of IL-12 in proliferation of various Th cell clones (class II-alloreactive (4-86 and 4-55) and keyhole limpet hemocyanin + self I-Ek-reactive (9-16)) following stimulation with Ag on APCs. These clones proliferated in response to stimulation with rIL-2, rIL-12, or Ag/APC. The proliferation induced by Ag/APC stimulation was not affected by anti-IL-2 Ab but was markedly inhibited by anti-IL-12 Abs. Consistent with this finding was the absence of detectable IL-2 activity in culture supernatants 12 to 48 h after Ag/APC stimulation, and the detection of significant levels of IL-12 in an Ab-capture bioassay. IL-12 was produced within 12 h after Ag/APC stimulation, reaching a peak after 18 to 24 h. The production of IL-12 in cultures of Th clones and APC contrasted with the production of IL-2 but not IL-12 upon allostimulation of primary T cells and the inhibition of their proliferation exclusively by anti-IL-2 Abs. Analysis of the expression of IL-12-binding sites on Th cells revealed low levels of IL-12 receptors in resting Th clones but high IL-12R levels 2 to 3 days after Ag/APC stimulation, declining gradually thereafter. The changes in IL-12R expression levels correlated closely with the IL-12 responsiveness of Th populations at various times after Ag/APC stimulation; Th populations obtained 3 and 10 days after Ag/APC stimulation exhibited very high and weak or marginal responsiveness to rIL-12, respectively, whereas the responses to rIL-2 were comparable in both Th populations. These results indicate that the Ag/APC-stimulated proliferation of terminally differentiated Th clones, in contrast to naive T cells, depends on the production of IL-12 by APC and on the simultaneous up-regulation of IL-12R on Th cells rather than on an IL-2 autocrine mechanism.

Animals↗
PubMed1996

Neutralization of IL-12 in vivo prevents induction of contact hypersensitivity and induces hapten-specific tolerance.

IL-12 is a heterodimeric cytokine with a powerful positive regulatory influence on the development of Th1 cell-mediated immune responses. Th1 cells are critically involved in contact hypersensitivity (CHS), so we were interested in studying whether IL-12 is of relevance in the induction phase of CHS. BALB/c mice were sensitized by epicutaneous application of 0.5% 2,4-dinitrofluorobenzene (DNFB) on the abdomen, draining lymph nodes obtained at various time points thereafter. RNA was extracted, and PCR analysis was performed using primers specific for the IL-12 subunits p35 and p40. Although p35 was constitutively expressed and not affected by hapten application, p40 transcripts were found to be enhanced in lymph nodes obtained between 12 and 24 h after sensitization. To study whether IL-12 is functionally relevant for the induction of CHS, an mAb directed against recombinant murine IL-12 was injected i.p. 3 and 24 h before sensitization. Challenge was performed 5 days later by painting the left ear with 0.3% DNFB. Whereas mice injected with sodium chloride or an isotype control Ab responded with a normal ear swelling after challenge with DNFB, mice treated with anti-IL-12 Ab showed a significantly reduced ear swelling response. To study whether injection of the IL-12 Ab causes transient nonresponsiveness or tolerance, resensitization was performed after a resting period of 14 days. Groups of mice initially treated with the anti-IL-12 Ab plus DNFB showed only a minimal response to DNFB after resensitization, suggesting that these mice became tolerant. Tolerance appeared to be hapten-specific because these mice could be successfully sensitized with the nonrelated hapten 2,4,6-trinitrochlorobenzene. Furthermore, when the anti-IL-12 Ab was injected into already sensitized mice before challenge, ear swelling response was significantly suppressed, suggesting that IL-12 also is involved in the effector phase of CHS. To exclude the possibility that the inhibitory effect of the anti-IL-12 Ab is simply due to an anti-inflammatory activity of the Ab, the anti-IL-12 Ab was injected i.p. 3 and 24 h before epicutaneous application of the toxic compound croton oil. However, irritant dermatitis elicited by croton oil was not affected by the Ab. Thus, the present study demonstrates that in vivo application of an anti-IL-12 Ab inhibits sensitization and induces hapten-specific tolerance and thus suggests that IL-12 may play a dominant in vivo role in the induction of CHS.

Animals↗
PubMed1996

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

Animals↗
PubMed1996

Biochemical characterization and protein kinase C dependency of monokine-inducing activities of Toxoplasma gondii.

Previous reports have indicated that the early induction of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), IL-1beta, and IL-10 is crucial for the establishment and regulation of host cell-mediated immunity to the intracellular protozoan parasite Toxoplasma gondii. In this study, we demonstrate that a soluble tachyzoite extract (soluble tachyzoite antigen) can trigger the expression of these four monokines by murine inflammatory macrophages. Further characterization revealed that the parasite molecules in soluble tachyzoite antigen responsible for monokine induction are heat stable at 100 degree C but differ in sensitivity to protease digestion. Thus, the tachyzoite factors that stimulate TNF-alpha and IL-to expression were found to be more resistant to treatment with proteinase K than those responsible for IL-12 and IL-10 induction. Similarly, while the factors responsible for the induction of all four monokines were found to be sensitive to periodate oxidation, the TNF-alpha-stimulating activity was partially resistant to treatment with the compound at a low concentration (1 mM). A further dichotomy in monokine induction signals was inferred from experiments with isoquinoline sulfonamide protein kinase inhibitors. The latter work suggested that the pathways for TNF-alpha and IL-1beta are protein kinase C dependent, while expression of IL-12 and expression of IL-10 share distinct signal transduction mechanisms involving other kinases. Together, these data argue that monokine induction by T. gondii is mediated by glycoproteins that may belong to distinct groups in terms of their biochemical properties and intracellular signaling pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

B7-1 and interleukin 12 synergistically induce effective antitumor immunity.

Enhanced host rejection of tumor cells is the primary goal of cancer immunotherapy and, in many murine tumor models, has been accomplished by engineering cells to express B7 costimulatory molecules or creating an environment rich in certain cytokines. We examined the effect of tumor cell B7-1 expression and administered recombinant interleukin 12 (IL-12) on the syngeneic host response to rapidly growing, poorly immunogenic SCK mammary carcinoma cells and to more slowly growing, immunogenic K1735 melanoma cells. Whereas B7-1 expression induced rejection of K1735 cells in 78% of mice, and IL-12 induced rejection in 38%, B7-1 expression induced rejection of SCK cells in only 28% of mice, and IL-12 induced rejection in none. The relative ineffectiveness of either B7-1 or IL-12 alone to induce rejection of SCK cells led us to combine the two manipulations. This resulted in rejection of SCK cells in 74% of mice and dramatically delayed tumor development in the remainder. Tumor rechallenge studies indicated that the surviving mice developed specific immunity to wild-type SCK cells. Lymphocyte subset ablation and IFN-gamma depletion studies indicated that rejection of SCK tumor cells brought about by the synergistic effects of B7-1 and IL-12 is mediated by a rapidly developing, systemic antitumor immune response that is dependent on the presence of both CD8+ and CD4+ T cells and involves IFN-gamma. Additionally, the synergistic effect of B7-1 expression and IL-12 administration is capable of inducing rejection of control SCK tumors simultaneously established in the opposite flank. The efficacy of B7-1 and IL-12 in inducing protective immunity against a poorly immunogenic, aggressive murine tumor indicates that this combination is particularly effective at producing a potent antitumor immune response that may be of therapeutic benefit.

Animals↗