In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor.

CD28 is a costimulatory receptor found on the surface of most T lymphocytes. Engagement of CD28 induces interleukin 2 (IL-2) production and cell proliferation when combined with an additional signal such as treatment with phorbol ester, an activator of protein kinase C. Recent studies have established that after CD28 ligation, the cytoplasmic domain of CD28 can bind to the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3 kinase). There is a concomitant increase in PI3 lipid kinase activity that may be important in CD28 signaling. Despite the requirement of phorbol 12-myristate 13-acetate (PMA) for effector function, we have found, however, that treatment of Jurkat T cells with the phorbol ester PMA dramatically inhibits (i) the association of PI3 kinase with CD28, (ii) the ability of p85 PI3 kinase to be immunoprecipitated by anti-phosphotyrosine antibodies, and (iii) the induction of PI3 kinase activity after stimulation of the cells with the anti-CD28 monoclonal antibody 9.3. These changes occur within minutes of PMA treatment and are persistent. In addition, we have found that wortmannin, a potent inhibitor of PI3 kinase, does not interfere with the induction of IL-2 after stimulation of Jurkat T cells with anti-CD28 monoclonal antibody and PMA. We conclude that PI3 kinase activity may not be required for CD28-dependent IL-2 production from Jurkat T cells in the presence of PMA.

DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans.

Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group- and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.