[Benefits and risks of immunostimulation with interleukin 12].
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Autoimmunity↗
Biomedical subjects
B Ryffel
Publications and source records attributed to B Ryffel.
Unanticipated human toxicology of recombinant proteins.
Recombinant human proteins play already an important role in therapy, e.g. erythropoietin and colony stimulating factors, while several promising candidates such IL-6, IL-12, thrombopoietin and others are in clinical development. Since the recombinant proteins are copies of endogenous proteins, it was assumed that they would be well tolerated. While this assumption is correct for some, other proteins proved to be a highly toxic. Therefore, preclinical safety assessment of these proteins is necessary. Based on the experience with several proteins, some guidance for the safety assessment can be given. Furthermore, data are presented demonstrating that preclinical toxicity studies have a predictive value for man. Limitations of the classical approach of safety tests and new concepts are discussed.
Animals↗
Effect of rapamycin on the expression of the IL-2 receptor (CD25).
The immunosuppressive macrolide rapamycin inhibits cytokine-driven proliferation of lymphocytes, acting at a later stage of T lymphocyte activation than the related compound FK506 or cyclosporin, which block IL-2 transcription. However, the effect of rapamycin on the expression of the IL-2 receptor alpha-chain (CD25) is less well documented. This study has investigated the effect of rapamycin on mRNA levels of CD25 and membrane expression of IL-2 receptor in human primary T lymphocytes activated by various stimuli. Rapamycin surprisingly inhibits CD25 upregulation subsequent to anti-CD3 or ionomycin stimulation. These effects are not secondary to an IL-2-mediated CD25 up-regulation, as rapamycin inhibits neither IL-2 synthesis nor IL-2-induced CD25 mRNA. Interestingly, sensitivity to rapamycin correlates with the requirement of de novo protein synthesis, as demonstrated by anisomycin inhibition of both ionomycin- and CD3-induced CD25 transcription. Thus, rapamycin inhibition of T cell activation may involve not only IL-2-driven proliferation, but also suppression of CD25 up-regulation.
Cells, Cultured↗
Gene knockout mice as investigative tools in pathophysiology.
An overview is given of the phenotype of mice with distinct deletions of genes for cytokines and some related proteins, which occur either naturally or as a result of homologous recombination. The use of such knockout mice for investigation of the pathophysiology of experimental disease is summarized. Understanding of the role of a given cytokine in pathogenesis has therapeutic implications, as the pathology may be prevented or attenuated by inhibition of the synthesis, release or activity of that cytokine.
Acute-Phase Proteins↗
Multiple immune abnormalities in tumor necrosis factor and lymphotoxin-alpha double-deficient mice.
To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-alpha in the development and function of the immune system, the Tnf and Ltalpha genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of IgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal lg levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vesicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.
Animals↗
Correction or transfer of immunodeficiency due to TNF-LT alpha deletion by bone marrow transplantation.
BACKGROUND: Mice with inactivated tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) genes have profound abnormalities of the immune system including lymphocytosis, lack of lymph nodes, undifferentiated spleen, hypoimmunoglobulinaemia, and defective Ig class switch. Here, we asked whether this phenotype is due to incompetent lymphohemopoietic progenitors or to a defective environment. MATERIALS AND METHODS: Lethally irradiated TNF-LT alpha-deficient and wild-type mice received bone marrow cells from either TNF-LT alpha-deficient or wild-type mice. The reconstitution and transfer of the phenotype was followed by morphological and functional analyses. RESULTS: Bone marrow cells from wild-type mice restored the synthesis of TNF and LT alpha, corrected the splenic microarchitecture, normalized the lymphocyte counts in the circulation, and repopulated the lamina propria with IgA-producing plasma cells of TNF-LT alpha-deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell dependent antigen was corrected, while no lymph nodes were formed. Conversely, the TNF-LT alpha phenotype could be transferred to wild-type mice by bone marrow transplantation after lethal irradiation. CONCLUSIONS: These data demonstrate that most TNF- and LT alpha-producing cells are bone marrow derived and radiosensitive, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow cell transplantation. The genotype of the donor bone marrow cells determines the functional and structural phenotype of the TNF-LT alpha-deficient adult murine host, with the exception of lymph node formation. These findings may have therapeutic implications for the restoration of genetically defined immunodeficiencies in humans.
Animals↗
Biochemical and morphological characterization of vascular and lymphocytic interleukin-4 receptors.
The distribution of the interleukin (IL)-4 receptor in normal human and common marmoset (Callithrix jacchus) tissues was examined by immunofluorescence and flow cytometry using monoclonal antibodies specific for the human IL-4 receptor to gain further insight into IL-4-mediated inflammatory and immunological events. IL-4 receptor positivity was unequivocally demonstrated on lymphocytes, predominantly T cells, and on blood vessels in many tissues. Vascular IL-4 receptor immunofluorescence consisted of a strong smooth muscle cell positivity and weaker positive staining of capillary and venular endothelial cells. Subnanomolar concentrations of IL-4 induced a genistein-sensitive up-regulation of VCAM-1 in vascular cell cultures. Tumor necrosis factor-alpha induced a genistein-resistant up-regulation of VCAM-1. IL-4 strongly induced expression of the IL-4 receptor on splenocytes (T lymphocytes) but not on vascular smooth muscle or endothelial cell cultures. Receptor cross-linking to [125I]IL-4 revealed a 65- to 75-kDa accessory receptor subunit consistent with a recently cloned IL-13 receptor associated with the IL-4 receptor on both vascular endothelial and smooth muscle cells. The demonstration of a vascular distribution pattern for the IL-4 receptor in addition to expression on lymphocytes suggests that vascular functional alterations, transduced through a unique IL-4 receptor complex (the type II IL-4 receptor), may be of importance during immunological and allergic inflammatory events.
Animals↗
Interferon-gamma enhances tumor necrosis factor-alpha production by inhibiting early phase interleukin-10 transcription.
The ability of cytokine synthesis inhibitory factor or interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) to modulate the production of tumor necrosis factor (TNF-alpha) induced by lipopolysaccharide (LPS) was examined in mouse bone marrow-derived macrophages (BMDM). IFN-gamma profoundly enhances LPS-stimulated TNF-alpha production, whereas IL-10 is markedly inhibitory, demonstrating the opposing effects of IFN-gamma and IL-10 on BMDM. Early neutralization of endogenously produced, LPS-stimulated IL-10 markedly enhanced short term TNF-alpha production, an effect further amplified by the absence of IFN-gamma priming. The regulatory effects of IFN-gamma and IL-10 apparently occurred at the translational (or post-translational) level, with TNF-alpha mRNA steady-state levels remaining unchanged. Furthermore, IFN-gamma exerts its enhancing effect on TNF synthesis by the transcriptional inhibition of IL-10. This in vitro finding was also confirmed in vivo. In the absence of LPS, IFN-gamma was not capable of inducing TNF-alpha production in BMDM, indicating that LPS or other signals are necessary for transcriptional activation. Reduced but significant TNF-alpha production in LPS-injected IFN-gamma receptor -/- mice suggests that IFN-gamma is not an absolute requirement and that other cytokines or cell types contribute in a secondary fashion to the priming of LPS-induced TNF-alpha production in vivo.
Animals↗
Cytokine knockout mice: possible application in toxicological research.
Knockout mice obtained by homologous recombination technology may be valuable tools for in vivo investigations in toxico-pathogenesis. A short review of the phenotype of mice with distinct cytokine deletions, which occur either naturally or are obtained by homologous recombination, is given. The possible application in pharmacological and toxicological research is discussed with examples of endotoxic shock, hepatic, renal and haematological toxicity. Further applications include a wide variety of cutaneous, pulmonary, vascular, infectious and autoimmune pathology.
Animals↗
Role of interferon-gamma in contact hypersensitivity assessed in interferon-gamma receptor-deficient mice.
The role of interferon-gamma (IFN gamma) in contact hypersensitivity induced by the haptens, oxazolone and 2,4,6-trinitrochlorobenzene (TNCB), was investigated in mice with a targeted disruption of the IFN gamma receptor (IFN gamma-R-/-). The 24-h ear-swelling response to oxazolone or TNCB in sensitized animals was not significantly reduced by the disruption of IFN gamma signalling. Dermal mononuclear infiltrates (MN) and epidermal microabscesses, however, were clearly diminished in the mutant mice. The hapten-induced upregulation of intercellular-adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I in IFN gamma-R-/- mice was smaller when compared to wild-type mice. It is concluded that oxazolone- and TNCB-induced contact hypersensitivity is partially dependent on a functional IFN gamma system. While the cutaneous oedema is IFN gamma-independent, the mononuclear cell infiltration and epidermal microabscess formation are at least partly IFN gamma-dependent. Therefore, reduced cellular infiltrates are likely due to a reduced upregulation of ICAM-1 and class I antigen expression in IFN gamma-R-/- mice.
Adjuvants, Immunologic↗
Distribution and activity of calcineurin in rat tissues. Evidence for post-transcriptional regulation of testis-specific calcineurin B.
Calcineurin (CN), a Ca2+/calmodulin-regulated phosphatase 2B, plays an important role in many biological processes including T-cell signal transduction. In the present study, the distribution and activity of CN were investigated in rat tissues. CN has a wide tissue distribution, as measured by enzyme-linked immunosorbent assay. CN concentrations are 0.2-0.6 micrograms/mg protein in most tissues, while the brain contains 3-10-fold higher concentrations. Immunohistochemical analyses using a monoclonal antibody to CN B subunit reveals that CN is not evenly distributed but concentrated in specific cells, especially in the brain, kidneys and testis. The specific enzymic activity of CN in tissues is around 10 pmol.min.mg protein-1, except in brain and liver (60 pmol.min-1.mg protein-1 compared to 3.6 pmol.min-1.mg protein-1). The immunosuppressants cyclosporin A and tacrolimus, but not rapamycin, inhibit the phosphatase activity of CN derived from most tissues tested, while CN activity from liver was resistant to cyclosporin A. Furthermore, transcripts and protein of the common CN B subunit and of the testis-specific form of CN B subunit were analyzed. The common CN B subunit transcripts and protein are detected in all tissues. Transcripts for the 'testis-specific' CN B subunit are also found in brain, lung, thymus and heart, while the protein is only detected in testis. This indicates that the testis-specific CN B subunit gene expression is regulated at both transcriptional and posttranscriptional levels. The findings demonstrate that CN is a widely distributed protein phosphatase and that its activity is regulated in a tissue-specific manner.
Amino Acid Sequence↗
The stimulatory effects of interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced interferon gamma in vivo.
Interleukin (IL)-12 synergizes with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors in vitro. However, in vivo administration of IL-12 decreases peripheral blood counts and bone marrow hematopoiesis. Here, we used interferon (IFN) gamma receptor-deficient (IFN gamma R-/-) mice to investigate whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 administered for 4 d (1 microgram/mouse per day) resulted in lower peripheral blood counts and a 2-fold decrease in bone marrow cellularity in wild-type mice, but not in IFN gamma R-/- mice. Bone marrow hematopoietic progenitors were decreased after IL-12 treatment in wild-type mice, but rather increased in IFN gamma R-/- mice. Splenic cellularity was 2.3-fold higher after IL-12 administration in wild-type mice, largely due to natural killer (NK) cell and macrophage infiltration together with some extramedullary hematopoiesis. In IFN gamma R-/- mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis. Thus, alterations mediated by IL-12-induced IFN-gamma include reduction in bone marrow cellularity and hematopoietic progenitors, as well as pronounced splenomegaly, largely caused by NK cell infiltration. In the absence of IFN-gamma signaling, IL-12 promotes hematopoiesis, consistent with its in vitro activities.
Animals↗
Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis.
Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-gamma receptor (sIFN-gamma R), anti-IFN-gamma monoclonal antibody (mAb) or IFN-gamma. All mice treated with sIFN-gamma R or anti-IFN-gamma mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-gamma-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-gamma- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-gamma R or anti-IFN-gamma mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-gamma inhibitor, such as the soluble IFN-gamma R, can be used for SLE therapy in the early stages of the disease.
Animals↗
Interleukin-2 receptor (CD25) upregulation on human T-lymphocytes: sensitivity to immunosuppressants is defined by the mode of T-lymphocyte activation.
We investigated the transcription and expression of the interleukin-2 receptor (IL-2R, CD25) in human T-lymphocytes after different modes of T-lymphocyte stimulation in the presence of the immunosuppressants cyclosporin (CsA) and tacrolimus (FK506) as well as the structurally related macrolide rapamycin. We demonstrate that CsA and FK506 inhibited IL-2R (CD25) gene transcription and protein expression after stimulation by anti-CD3 or ionomycin, but not by phorbol ester or IL-2. Rapamycin, which does not affect IL-2 transcription, surprisingly inhibited IL-2R upregulation after anti-CD3- or ionomycin-induced stimulation, but not by phorbol ester or IL-2. Interestingly, the protein synthesis inhibitor anisomycin inhibited anti-CD3- or ionomycin-, but not phorbol ester- and IL-2-induced CD25 gene transcription indicating the dependence on de novo protein synthesis of the former pathways. These data suggest that the pharmacological effect of the imunosuppressants tested on IL-2R gene expression may be predicted by the requirement of protein synthesis of a given activation pathway.
Cells, Cultured↗
Role of proinflammatory cytokines in a toxic response: application of cytokine knockout mice in toxicological research.
The toxic effect of xenobiotics may be direct or mediated through the release of secondary toxic mediators such as proinflammatory cytokines. Knockout mice (KO or -/-) with discrete gene deletions obtained by homologous recombination may prove the function of a given cytokine in general and in toxico-pathogenesis. The technology is briefly described, and a short review of the phenotype of mice with deletions of several proinflammatory cytokines is given. The emphasis of this presentation is on the role of tumor necrosis factor, interleukins and interferon-gamma in endotoxic shock and acute phase response. The future application of these mice for mechanistic studies in toxicological research is discussed.
Acute-Phase Reaction↗
MHC antigens in interferon gamma (IFN gamma) receptor deficient mice: IFN gamma-dependent up-regulation of MHC class II in renal tubules.
MHC class II gene products in parenchymal cells, such as tubular epithelial cells in kidney, may play a role in the regulation of autoimmune reactions. Expression of MHC class II in renal tubular cells is normally very low, but it increases considerably under various pathologic conditions. The predominant role of IFN gamma in up-regulation of MHC class II expression has been demonstrated repeatedly. We tested the existence of alternative pathways of MHC class II regulation using IFN gamma receptor-deficient (IFN gamma R-/-) mice. Mutant and wild type mice received 50 micrograms bacterial endotoxin (LPS) i.p. Four days later the kidneys were removed for immunofluorescence examination. In agreement with published results LPS provoked an increase of immunoreactivity for MHC class I and MHC class II in proximal tubules of wild type mice. While MHC class I up-regulation was strictly IFN gamma receptor-dependent, up-regulation of MHC II was still evident in mutant mice, although less than in wild type mice. Since injection of IFN gamma induced proximal tubular MHC class II expression in wild type mice but not in IFN gamma R-/- mice, an alternative signaling pathway for IFN gamma does not seem to exist. Thus, up-regulation of MHC class II expression in renal tubules does not necessarily require IFN gamma. The markedly patchy pattern of immunofluorescence in IFN gamma R-/- mice suggests that induction of MHC class II after LPS injection may represent renal injury due to shock.
Animals↗
In situ detection of cyclosporin A: evidence for nuclear localization of cyclosporine and cyclophilins.
BACKGROUND: The immunosuppressant cyclosporin A (CsA) forms a trimolecular complex with cyclophilin (CPH) and calcineurins (CN) and inhibits CN phosphatase activity. Inhibition of CN phosphatase by CsA prevents the dephosphorylation of a nuclear factor in the cytosol and its nuclear translocation to the nucleus. EXPERIMENTAL DESIGN: The intracellular distribution of CPH and CN was investigated in permeabilized Jurkat T lymphocytes and MRC fibroblasts using biochemical techniques and confocal microscopy. The site of CsA binding was identified in situ using a photoaffinity label derivative of CsA followed by immunodetection. RESULTS: Cyclophilin A (CPH-A) and CN display essentially a cytosolic localization by immunofluorescence, and additional nuclear CPH-A and CN are evidenced by Western blot analysis of purified nuclei and immunofluorescence. By contrast, cyclophilin B (CPH-B) has a punctuate and reticular distribution pattern in cytoplasm, indicating an association with the endoplasmatic reticulum, but its main location is in the nuclear matrix, sparing the nucleolar region. For the in situ detection of CsA binding sites, a photolabile cyclosporine derivative (PL-CS) was used that allowed the detection of covalently bound CsA by Ab. Using the biologically active PL-CS, a punctate cytoplasmatic and nuclear immunoreactivity was obtained, which was specific and competed only with active cyclosporine derivatives. Nuclear CPH-A and -B were labeled by PL-CS, and trimolecular complexes of labeled CPH and CN were obtained by chemically cross-linking nuclear extracts. CONCLUSIONS: We describe herein the accessibility of CsA to the nucleus, the presence and labeling in situ of nuclear CPH and CN. The current models of CsA action predict that CsA-CPH complexes inhibit CN activity in the cytosol. However, our present findings invite the hypothesis that CPH may capture the drug into the nucleus and target regulatory proteins or transcriptional control elements.
Amino Acid Isomerases↗