Anneliese Schimpl

Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene

Mice deficient for interleukin-2 develop normally during the first 3-4 weeks of age. However, later on they become severely compromised, and about 50% of the animals die between 4 and 9 weeks after birth. Of the remaining mice, 100% develop an inflammatory bowel disease with striking clinical and histological similarity to ulcerative colitis in humans. The alterations of the immune system are characterized by a high number of activated T and B cells, elevated immunoglobulin secretion, anti-colon antibodies, and aberrant expression of class II major histocompatibility complex molecules. The data provide evidence for a primary role of the immune system in the etiology of ulcerative colitis and strongly suggest that the disease results from an abnormal immune response to a normal antigenic stimulus.

Control of T cell hyperactivation in IL-2-deficient mice by CD4+CD25– and CD4+CD25+ T cells: evidence for two distinct regulatory mechanisms

In IL‐2‐deficient mice, antigen‐activated CD4 T cells accumulate and cause lethal immune pathology. Wild‐type cells of hematopoietic origin present in the same animal are able to prevent this hyperactivation of T cells, but the mechanisms and cells controlling the IL‐2‐deficient cells are unknown. Here we show that IL‐2– CD4 cells with an ovalbumin‐specific transgenic TCR (IL‐2– OVAtg) undergo both clonal expansion and clonal contraction when transferred to euthymic recipients and challenged with antigen, but continuously expand in athymic hosts. Cotransfer of wild‐type CD4 T cells prevents the accumulation of IL‐2‐deficient cells. On the residual IL‐2– TCRtg cells CD69 and CD25 are up‐regulated, suggesting that activation per se is not suppressed and that the cells had received an IL‐2 signal. Since IL‐2 is able to restore the defective antigen‐induced cell death (AICD) of IL‐2‐deficient T cells in vitro, paracrine IL‐2 provided by the wild‐type CD4 cells may thus be able to allow clonal contraction of IL‐2‐deficient cells also in vivo. Interestingly however, regulatory CD4+CD25+ cells also efficiently contain the clone size of antigen‐stimulated IL‐2‐deficient T cells. Since CD4+CD25+ cells do not produce IL‐2, this suggests a mechanism of suppression distinct from paracrine IL‐2 delivery. In keeping with this, the residual IL‐2– TCRtg cells recovered after cotransfer of regulatory CD4+CD25+ cells do not show increased CD25 or CD69 expression, suggesting that they had not received paracrine IL‐2 and that clonal containment occurred at the level of initial activation rather than clonal contraction by AICD. IL‐2 deficiency therefore mayupset T cell homeostasis by two distinct mechanisms: the failure to program expanding T cells for apoptosis, and the failure to generate functional CD4+CD25+ regulatory cells.

Autoregulation of NFATc1/A Expression Facilitates Effector T Cells to Escape from Rapid Apoptosis

Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.

A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death

Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. We show here that cross‐linking CD40 stimulates the expression of A1, a member of the anti‐apoptotic Bcl‐2 family, in primary murine B lymphocytes. CD40‐dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line. We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein. A1‐transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor. In contrast, constitutive expression of A1 did not abrogate anti‐IgM‐induced c‐myc down‐regulation. Consistant with this, A1 did not release anti‐IgM‐induced cell cycle arrest. Our data indicate that CD40‐stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti‐IgM antibodies and suggests a protective role for A1 in antigen receptor‐mediated apoptosis in B cells.

IL-2 and autoimmune disease.

A decade after the first description of IL-2-deficient mice, the redundancy of IL-2 as a T cell growth factor is well accepted and the focus of research has shifted to the unexpected multiorgan autoimmunity and inflammation observed in mice lacking components of the IL-2/IL-2R system. So far, a set of defects at the levels of repertoire selection, the generation of suppressive regulatory T cells, T cell homing and clonal contraction via activation induced cell death (AICD) have been documented. We propose that these individual defects jointly contribute to the severe disturbance of T cell homeostasis and self-tolerance underlying the immunopathology of the IL-2 deficiency syndrome.

Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice

NF‐ATp and NF‐ATc are the most prominent nuclear NF‐AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF‐ATp in vivo we have inactivated the NF‐ATp gene by gene targeting in mice. We show that NF‐ATp deficiency leads to the accumulation of peripheral T cells with a “preactivated” phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen‐reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF‐ATp‐deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF‐ATp‐deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+ /IgD+ B lymphocytes. Contrary to the T lymphocytes from IL‐2‐deficient mice which develop a phenotype similar to the NF‐ATp− / − mice, NF‐ATp− / − T cells do not show obvious defects in Fas‐mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF‐ATp.

REVERSAL OF BLIMP-1-MEDIATED APOPTOSIS BY A1, A MEMBER OF THE BCL-2 FAMILY

Blimp‐1 (B lymphocyte‐induced maturation protein 1) is strongly expressed during the late stages of B cell differentiation to immunoglobulin‐secreting plasma cells. Overexpression of Blimp‐1 in B lymphoma cells has been reported to induce either growth arrest and cell death or Ig secretion and terminal differentiation, depending on the developmental stage of the recipient lymphomas. By using a retroviral expression system we show that Blimp‐1‐transduced immature WEHI 231 murine B lymphoma cells produce J chain, increased levels of the secretory form of μ heavy chain mRNA and secrete IgM for a short period of time. Concomitantly, they exhibit altered ratios of c‐myc/mad4 mRNA levels, a reduction in the expression of the anti‐apoptotic bcl‐2 family member A1 and a distinct growth disadvantage, followed by cell death. Reintroduction of A1 by retroviral transduction greatly extends the life span of Blimp‐1‐expressing WEHI 231 cells which continue to secrete IgM. These data suggest that levels of A1 may determine the checkpoint between death and survival of Blimp‐1‐expressing B cells at different stages of differentiation.

A third signal in b cell activation given by trf.

There is a wealth of experimental evidence supporting the idea that T cells have to intervene in the immune response to many antigens to ensure an optimal antibody response (Reviewed in Greaves et al. 1973). We have addressed our work to the question at whicli step of B cell activation this intervention is necessary. An in vitro culture system (Mishell & Dutton 1967) was applied using spleen cells of thie functionally athymic nu/nu mice and heterologous blood cells as antigens. SRBC can operationally be defined as T cell dependent antigens since the IgM immune response in T cell deprived cultures is far from the optimal response obtained in the presence of T cells (Schimpl & Wecker 1970). In an attempt to restore the immune response in T cell deprived cultures with thymocytes we found that allogeneic thymocytes were greatly superior to syngeneic ones (Schimpl & Wecker 1971). Subsequent experiments showed that the effect could be ascribed to a soluble factor produced by allogeneically or Concanavalin A (Con A) stimulated T cells (Schimpl & Wecker 1972, Wecker et al. 1974). Soluble mediators with similar T cell replacing activities have now been described by many different groups (Dutton et al. 1971, Doria et al. 1972, Gorczynski et al. 1972, Feldmann & Basten 1972, Britton 1972, Sjoberg et al. 1972, Gisler et al. 1973, Rubin et al. 1973, Waldmann & Munro 1973, Watson 1973, Kishimoto & Ishizaka 1973, Amerding & Katz 1974), some factors being obtained from T cells, activated by antigen in vivo and incubated with the same antigen in vitro. The assumption imderlying all further considerations is that such mediators are regularly produced upon stimulation of T cells and that they

Protein Kinase A Regulates GATA-3-Dependent Activation of IL-5 Gene Expression in Th2 Cells

Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4+ T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit α enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.

C/EBPβ enhances IL‐4 but impairs IL‐2 and IFN‐γ induction in T cells

C/EBP transcription factors have been described to control the activity of the human IL‐4 promoter. The C/EBP binding sites within the IL‐4 promoter overlap with composite NF‐AT and AP‐1 binding motifs. We show here that similar binding sites are part of the murine IL‐4 promoter. Retroviral overexpression of C/EBPβ in murine EL‐4 thymoma cells led to a strong induction of endogenous IL‐4 and a reduction in IL‐2 and IFN‐γ expression. Similarily, in primary murine T cells C/EBPβ induction resulted in an increase in IL‐4 levels, whereas in human Jurkat T cells a decrease in IL‐2 RNA was detected. Like AP‐1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP‐1, C/EBPβ does not act in synergy with NF‐AT in the induction of the murine IL‐4 promoter. Instead, both factors compete in their binding to the P4/Pu‐bD site, one of the most important sequence elements of the IL‐4 promoter. Whereas NF‐AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL‐4 promoter activity at specific developmental stages of T cells.