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Biomedical subjects

G Kroemer

Publications and source records attributed to G Kroemer.

At least 19 recordsLinked to original sources
PubMed1992

Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.

Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.

Animals↗
PubMed1992

Cross-linking of surface IgM or IgD causes differential biological effects in spite of overlap in tyrosine (de)phosphorylation profile.

Although displaying similar amounts of surface IgM and IgD, ECH 408-1 cells only succumb to apoptosis after cross-linking of IgM (not IgD), suggesting that different signaling pathways couple to both receptors. Immunoprecipitation studies revealed the presence of several proteins selectively associated with IgM and IgD, thus ruling out that the lack of inhibitory signaling mediated by IgD might be due to membrane expression in the absence of associated proteins belonging to the B cell receptor complex. 32P metabolic labeling and immunoprecipitation studies demonstrated that IgM and IgD are associated with phosphoproteins of 32-33 kDa in an isotype-specific fashion. Kinetic analyses of tyrosine kinase activity showed that cross-linking of surface IgM or IgD resulted in the rapid (1-3 min) phosphorylation of several protein substrates on tyrosine residues, followed by a dephosphorylation step. Isotype-specific changes of the phosphorylation status specifically affected molecules in the 32-33 kDa range, i.e. IgM (not IgD) cross-linking affected a approximately 32-kDa protein, whereas IgD (not IgM) cross-linking induced phosphorylation of a protein exhibiting a slightly lower mobility (33 kDa). These results suggest that isotype-specific immunoglobulin-associated molecules could be involved in the second messenger cascade leading to different biological effects upon IgM and IgD cross-linking.

Animals↗
PubMed1992

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 beta 8 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 beta 8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing "anergy" and apoptotic cell death of V beta 8+ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V beta 8+ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)alpha chains in vivo, although SEB induces IL-2R alpha 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 beta 8+ 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.

Animals↗
PubMed1992

lpr T cells vaccinate against lupus in MRL/lpr mice.

MRL/MP-lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8-) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease-accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp-lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This "T cell vaccination" approach resulted in a selective depletion of cells expressing products of the V beta 8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.

Animals↗
PubMed1992

Attenuation of autoimmune disease and lymphocyte accumulation in MRL/lpr mice by treatment with anti-V beta 8 antibodies.

MRL-MP-lpr/lpr mice are afflicted by a severe systemic autoimmune disease that is aggravated by the lpr mutation resulting in the accumulation of phenotypically abnormal lpr cells (CD3+CD4-CD8-) in all lymphoid issues including hyperplastic lymph nodes. Given that products of the T cell receptor V beta 8 gene family are overrepresented among lpr cells, different schedules aimed at selectively decreasing the frequency of lpr cells were designed. First, continuous administration of the monoclonal antibody F23.1 (specific for V beta 8 products) resulted in a significant depletion of V beta 8+ cells and prevented the manifestation of lymph accumulation at the same time as it reduced the serological, clinical, and histopathological signs of autoimmune disease. Along the same line, administration of either F23.1 or two different anti-F23.1 anti-idiotypic antibodies to MRL/Mp-lpr/lpr mothers elicited, in the offspring, the production of antibodies sharing a recurrent idiotype with F23.1 and resulted in long-term amelioration of autoimmunity and lymphadenopathy. Thus, a strategy aimed at specifically reducing the frequency of a subset of lpr cells proved successful in mitigating the autoimmune process.

Animals↗
PubMed1992

Inability of IL-2 and IL-10 to counteract B cell clonal deletion.

The B cell antigen receptor (BCR) delivers inhibitory signals in nascent B cells leading to the establishment of tolerance via clonal deletion or clonal anergy depending upon the type of antigen to which the B cells are exposed. In previous work, it has been demonstrated that activated Th2 cells, as well as some recombinant lymphokines, prevent the inhibition of growth and subsequent cell death induced through the BCR in model B cell lymphomas. Herein, we extend this work to another Th2 lymphokine, IL-10, that in contrast to IL-4 does not interfere with the deletion promoted by IgM crosslinking. The effect of individual lymphokines has also begun to be analyzed in a transgenic model of B cell clonal deletion. To this end, we have administered a recombinant vaccinia virus producing human IL-2 to mice expressing an autoreactive H-2Kk,b-specific transgenic IgMk and found that IL-2 does not abrogate B cell deletion in vivo.

Animals↗
PubMed1992

Interferon induction of chicken MHC class I gene expression: phylogenetic conservation of the interferon-responsive element.

The 5' upstream region of a chicken MHC class I gene BF-IV contains sequence motifs similar to the interferon consensus sequences (ICS) contained in promoters of many mammalian interferon-regulated genes. To study a possible functional role of this putative chicken ICS, an oligonucleotide spanning the upstream sequences of the BF-IV gene (-174/-194) was cloned singly or in multiple copies before the herpes TK promoter controlling the chloramphenicol acetyl transferase (CAT) gene (pBLCAT2). Transient expression studies performed with primary chicken fibroblasts (CEF) showed that the chicken ICS represses constitutive promoter activity. The chicken ICS, however, enhanced CAT activity up to 20-fold following treatment with chicken interferon (IFN). Deletion analysis of the BF-IV promoter also confirms that the upstream DNA sequences (-174/-194) contain a functional ICS recognized by chicken interferon. The murine ICS of the H2-Ld gene was also activated by chicken interferon when introduced into CEF. IFN activation of chicken ICS containing reporters was also observed in transformed chicken fibroblast lines. We show that the chicken ICS binds two specific nuclear factors present in chicken fibroblasts which are induced by interferon. These factors were also capable of recognizing the mouse ICS, suggesting the conservation of a relevant DNA-binding protein. Taken together, these data indicate that the chicken ICS motif contained in a sequence from -174 to -194 of the BF-IV gene acts as a strong interferon-response element, which has been functionally conserved during about 270 million years of separate evolution of mammals and birds.

Animals↗
PubMed1992

Mechanisms of self tolerance.

The redundancy of biological systems minimizes the probability that isolated molecular and cellular defects entail deleterious consequences. This notion also applies to the establishment and maintenance of tolerance to self antigens. Thus, immune homeostasis is attributed to multiple distinct safety valves that are connected in series and intervene at defined control points of the life cycle of the developing lymphocyte to guarantee the physical elimination, functional inactivation, or regulated inhibition of self-reactive, potentially autoaggressive, B and T cells.

Animals↗
PubMed1992

Interleukin-2: a possible trigger for autoimmunity.

High doses of recombinant interleukin-2 (IL-2) may induce autoimmune lesions in patients receiving experimental cancer treatment. In most cases, the manifestation of autoaggression is transient and organ-specific, predominantly affecting the thyroid gland. Only a fraction of the patients are concerned; most individuals (around 90%) do not develop any signs of autoimmunity. Apparently, endogenously hyperproduced IL-2 may also be implicated in the pathogenesis of autoaggression, since active phases of such disparate autoimmune diseases, like multiple sclerosis and systemic lupus erythematosus, are accompanied by elevated IL-2 serum levels. Taking into account that immunological self-tolerance is maintained by several distinct mechanisms, we investigated whether IL-2 would interfere with clonal deletion or clonal anergy in vivo. In several experimental systems, IL-2 failed to abolish clonal deletion in the murine thymus or in the peripheral T-cell compartment. IL-2 did not affect the clonal deletion of self-reactive B cells in the bone marrow either. In contrast, IL-2 was found to be effective in abrogating clonal anergy of non-deleted self-specific T cells. Only in the presence of high frequencies of self-specific, potentially autoreactive T cells, IL-2 induces autoimmune lesions. Thus, IL-2 interferes with a mechanism of self-tolerance that guarantees the inactivation of T cells that for some reason have 'escaped' clonal deletion. If these data, obtained in the murine system, are extrapolated to man, then it may be stated that the T-cell repertoire of most individuals has been completely purged from self-reactive cells. Only in the presence of a non-deleted, anergic, potentially auto-reactive T-cell population, could organ-specific disease be induced by IL-2.

Animals↗
PubMed1992

Coevolution of hosts and microorganisms: an analysis of the involvement of cytokines in host-parasite interactions.

Parasites may employ particular strategies of eluding an immune response by taking advantage of those mechanisms that normally guarantee immunological self-tolerance. Much in the way as it occurs during the establishment of self-tolerance, live pathogens may induce clonal deletion, functional inactivation (anergy) and immunosuppression. At this latter level, it appears that certain pathogens produce immunosuppressive cytokine-like mediators or provoke the host to secrete cytokines that will compromise the anti-parasite immune response. It appears that immune responses that preferentially involve T helper 1 cells (secretors of interleukin-2-and interferon-gamma) tend to be protective, whereas T helper 2 cells (secretors of IL-4, IL-5, IL-6, and IL-10), a population that antagonizes T helper cells, mediate disease susceptibility and are involved in immunopathological reactions. Cytokines produced by T helper 2 cells mediate many symptoms of infection, including eosinophilia, mastocytosis, hyperimmunoglobulinemia, and elevated IgE levels. Administration of IL-2 and IFN-gamma has beneficial effects in many infections mediated by viruses, bacteria, and protozoa. The use of live vaccinia virus might be an avenue for the treatment of or the vaccination against infection. We have found that a vaccinia virus expressing the gene for human IL-2, though attenuated, precipitates autoimmune disease in immunodeficient, athymic mice. Thus, although T helper 1 cytokines may have desired immunostimulatory properties, they also may lead to unwarranted autoaggressive responses.

Animals↗
PubMed1992

The role of interleukin 2 in the development of autoimmune thyroiditis.

Interleukin 2 (IL-2) is a lymphokine that may disrupt immunological self-tolerance. While being incapable of interfering with intrathymic or peripheral clonal deletion, IL-2 may overcome functional antigen unresponsiveness in anergic T lymphocytes. Anergy of T helper cells of the inflammatory phenotype implies selective silencing of the transcription of the IL-2 gene and thus precludes autocrine IL-2/IL-2 receptor (IL-2R) mediated growth, as well as delivery of help to other T cells or B lymphocytes. Thus, IL-2 serves as a servomodulator regulating post-deletional self-tolerance. IL-2-producing and IL-2-receptive cells are present in a variety of autoimmune lesions, including spontaneous autoimmune thyroiditis developing in the Obese strain (OS) of chickens, in Hashimoto's struma lymphomatosa, and in Graves' disease. Whereas the OS is characterized by a hyperinducibility of the IL-2/IL-2R system that predisposes to the development of severe thyroid infiltration, the state of the IL-2/IL-R system in circulating lymphocytes of patients developing thyroid autoimmunity, or at risk of doing so, remains to be defined. The most frequent autoimmune side-effect of IL-2 treatment concerns the thyroid gland. IL-2 induces a lymphoid thyroiditis leading to primary hypothyroidism, especially in those patients that have pre-treatment antithyroid autoantibodies. The hypothesis is extrapolated that IL-2 induces autoimmune disease in those patients that bear undeleted thyroid-specific T cells, and in which the lack of manifest thyroiditis relies upon peripheral, post-deletional tolerance.

Animals↗
PubMed1992

Interleukin-2, a pro-autoimmune lymphokine that interferes with post-deletional tolerance.

Various complementary mechanisms arranged in a fail-safe hierarchy impede the immune system from launching auto-aggressive attacks, namely intrathymic or post-thymic clonal deletion, anergy, and immunosuppression. Both epidemiological and clinical evidence demonstrates that interleukin-2 (IL-2) may exert a pro-autoimmune effect. Thus, IL-2 reverses immunological tolerance in determined in vitro or in vivo circumstances, is produced at abnormally high levels in certain autoimmune diseases, and induces organ-specific autoimmune lesions when administered to patients. To unravel the molecule and cellular mechanisms responsible for the pro-autoimmune nature of IL-2, our group has employed a recombinant hIL-2 vaccinia virus construct (IL-2. VV) as a self-replicating IL-2-releasing device that may be administered as a source of exogenous IL-2 to experimentally manipulated mice. IL-2 does not interfere with clonal deletion of potentially autoreactive T cells in the thymus since application of IL-2. VV to young mice fails to augment the frequency of T cells bearing 'forbidden' T cell receptor (TCR) V beta gene products (i.e. T cells that recognize endogenous retroviral superantigens). Along the same line, IL-2. VV does not reverse the clonal deletion in male mice bearing a male-specific transgenic alpha/beta TCR that undergo depletion of CD4+ CD8+ thymocytes, nor does it abolish post-thymic clonal deletion of V beta 8+ T cells reactive with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB). In contrast with its incapability to abolish intra- and post-thymic elimination of T cells with unwarranted specificities, IL-2 abrogates anergy of T cells. Non-deleted, 'forbidden' T cells from congenitally athymic or neonatally thymectomized mice, under normal circumstances anergic, become responsive to TCR-triggering and mediate a systemic autoimmune syndrome upon IL-2. VV treatment. Thus, IL-2 appears to interfere with T cell tolerance at a post-deletional stage, reversing functional non-responsiveness and enabling non-deleted T cells that bear a potentially autoreactive TCR repertoire to cause manifest autoimmunity.

Animals↗
PubMed1991

The fail-safe paradigm of immunological self-tolerance.

Immunological self-tolerance is guaranteed by several complementary mechanisms arranged in a fail-safe hierarchy--namely, clonal deletion, inactivation, and suppression. Only failure of all these self-tolerance-preserving systems allows autoimmune disease to develop. The notion of such a fail-safe organisation of the immune system is compatible with several features of autoimmune disease: its multicausal nature, its slowly progressive and relapsing course, and the diverse range of interventions available to restore autotolerance. This hypothesis has practical implications for the understanding of both the pathogenesis of iatrogenic autoimmune disease and the design of strategies to re-establish self-tolerance.

Autoimmune Diseases↗
PubMed1991

Involvement of the interleukin 4 pathway in the generation of functional gamma delta T cells from human pro-T cells.

We have used the technique of in situ hybridization to investigate the transcription of genes encoding the CD3 complex and the lymphokine interleukin 4 (IL-4) by human pro-T cells--i.e., cells that phenotypically resemble those T-cell precursors that colonize the thymus during early intrathymic development. CD1-2-3-4-7+8-45+ pro-T cells isolated from postnatal thymi via immunoselection with a panel of specific monoclonal antibodies are already committed to the T-cell lineage because most of them transcribe the genes encoding the delta and epsilon chains of the CD3 complex. About half of such pro-T cells synthesize IL-4 mRNA in the absence of any exogenous stimulation. Upon culture with IL-4, pro-T cells extensively proliferate and differentiate into functionally competent, mature gamma delta T cells expressing a T-cell receptor repertoire similar to that of gamma delta T cells that can be found in postnatal thymus. The IL-4 response of pro-T cells is not mediated by induction of the interleukin 2 (IL-2)-IL-2 receptor pathway and, unlike IL-2-driven T-cell differentiation, does not require the presence of stromal cells. Taken altogether, these findings suggest that an autocrine IL-4-mediated pathway might be implicated in early thymocyte differentiation--namely, in the generation of T cells bearing the gamma delta T-cell receptor.

Antibodies, Monoclonal↗
PubMed1991

Interleukin 2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice.

Under physiological conditions, the vast majority of T cells differentiate in the thymus, an organ that provides an optimal microenvironment for T cell maturation and shapes the T cell repertoire via positive and negative selection processes. In the present report, we demonstrate that neonatal thymectomy of CBA/H mice results in a diminution of T cells in peripheral lymphoid organs (spleen, lymph nodes), but is followed by a marked transient (12 wk) increase in Thy-1+ CD3+ cells in the peritoneal cavity. These cells exhibit predominantly a double-negative (CD4-CD8-) phenotype among which products of the T cell receptor (TCR) V beta 11 gene family (i.e., an I-E-reactive TCR normally deleted in I-E-bearing CBA/H mice) are selectively overexpressed. This observation suggests that, under athymic conditions, T cell differentiation and/or accumulation may occur in the peritoneal cavity. Intraperitoneal inoculation of an interleukin 2 (IL-2) vaccinia virus construct that releases high titers of human IL-2 in vivo induces conversion of these double-negative T cells to either CD4+ CD8- or CD4- CD8+ single positives, and allows in vitro stimulation of TCR V beta 11-bearing cells with a clonotypic anti-V beta antibody. Since IL-2 induces autoimmune manifestations (DNA autoantibodies, rheumatoid factors, and interstitial nephritis) in thymectomized CBA/H mice, but not in sham-treated littermates, this lymphokine is likely to enhance the autoaggressive function of T cells that bear forbidden, potentially autoreactive TCR gene products and that are normally deleted in the thymus.

Animals↗
PubMed1991

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes.

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoire is a distorted continuum of homologous genes that may be grouped into families displaying greater than 80% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Igk-V families (sequence similarity generally greater than 74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comparisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.

Animals↗