The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A.

CD28 and CTLA-4 are related members of a family of T lymphocyte cell surface receptors that function to regulate T cell activation. We have found that the cytoplasmic domains of both CTLA-4 and CD28 can associate with members of the PP2A family of serine/threonine phosphatases. The association of PP2A with CD28 was negatively regulated by tyrosine phosphorylation of the CD28 cytoplasmic domain. Inhibition of PP2A activity in Jurkat leukemia T cells by treatment with okadaic acid or by expression of a dominant-negative mutant enhanced T cell activation induced by CD28 engagement. Interactions between cell surface receptors such as CTLA-4 and CD28 and serine/threonine phosphatases may represent a novel mechanism for modulating the intracellular signal transduction pathways associated with cell activation.

Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice null for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

Lineage-restricted function of nuclear factor kappaB-inducing kinase (NIK) in transducing signals via CD40.

CD40 signaling in B cells and dendritic cells (DCs) is critical for the development of humoral and cell-mediated immunity, respectively. Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) has been implicated as a central transducing kinase in CD40-dependent activation. Here, we show that although NIK is essential for B cell activation, it is dispensable for activation of DCs. Such data provide compelling evidence that different intermediary kinases are used by different cellular lineages to trigger NF-kappaB activation via CD40.

Activation and regulation of the IkappaB kinase in human B cells by CD40 signaling.

This study demonstrates that the engagement of CD40 results in the activation of the recently described IkappaB kinase (IKK) in a human B cell line. The kinase appears to reside within the cell in a cytosolic signalsome complex consisting of IKK, IkappaB, and an MKP-1-like molecule. While the binding of CD154 to CD40 induces the assembly of a CD40-TRAF receptor complex, IKK is not recruited to this complex. Nonetheless, a functional link between TRAF2 and IKK activity in B cells is demonstrated by the fact that overexpression of TRAF2 constitutively induces IKK activity, NF-kappaB luciferase and Fas expression. Synergy in the activation of IKK and NF-kappaB-dependent gene expression was observed by the simultaneous engagement of the B cell receptor and CD40, establishing an early means for cross-talk between these two B cell activation pathways. This study discusses the sequential biochemical events that transpire upon CD40 engagement by its ligand in human B cells.

Regulation of cytotoxic T lymphocyte-associated molecule-4 by Src kinases.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.

Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases.

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia.

Collagenase-3 (matrix metalloprotease 13) is preferentially localized in the deep layer of human arthritic cartilage in situ: in vitro mimicking effect by transforming growth factor beta.

OBJECTIVE: To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) on the synthesis and distribution of collagenase-3. METHODS: Human cartilage specimens were obtained from tibial plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-1beta (100 units/ml) or TGFbeta (150 ng/ml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody. RESULTS: The number of cells that stained for collagenase-3 in normal cartilage was very low (approximately 3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean +/- SEM 58.4 +/- 1.6% and 40.1 +/- 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 +/- 3.2% and 43.2 +/- 2.7%; P < 0.01). Similarly, RA cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 +/- 4.1%) compared with the superficial layers (26.2 +/- 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and at a distance from the pannus junction. Both IL-1beta and TGFbeta increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFbeta had a predominant effect in the deep layers, while IL-1beta had a greater effect on the superficial layers. CONCLUSION: This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFbeta can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.

Assembly and regulation of the CD40 receptor complex in human B cells.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. Studies with human B cells show that the binding of CD154 (gp39, CD40L) to CD40 recruits TNF receptor- associated factor 2 (TRAF2) and TRAF3 to the receptor complex, induces the downregulation of the nonreceptor-associated TRAFs in the cell and induces an increased expression of Fas on the cell surface. Combined signaling through the interluekin 4 receptor and CD40 induces an increased expression of Fas with a commensurate increase in the level of TRAF2, but not TRAF3, that is recruited to the receptor complex. In contrast, engagement of the membrane immunoglobulin and CD40 limits Fas upregulation and reduces the recruitment of TRAF2, relative to TRAF3, to the CD40 receptor complex. These studies show that the TRAF composition of the CD40 receptor complex can be altered by signals that influence B cell differentiation.

Cloning, sequencing and characterization of the 5'-flanking region of the human collagenase-3 gene.

Collagenase-3 (matrix metalloprotease-13) is a recently discovered human collagenase produced in normal articular cartilage chondrocytes and thought to be involved in the pathological process of osteoarthritis. We have sequenced and characterized 1.6 kb of the human collagenase-3 gene 5'-flanking region. The transcription start site was located 22 bp upstream from the ATG start codon. Sequence analysis of the 5'-flanking region revealed the presence of the consensus recognition sites for the TATA and CCAAT DNA-binding proteins, activator protein-1 and E26 transformation specific/polyoma virus enhancer, as well as three core motifs of hormone response elements. Transient transfection assays demonstrated that a small fragment of 133 bp, containing the activator protein-1 and E26 transformation specific/polyoma virus enhancer sites promoted transcription in normal and osteoarthritic human chondrocytes with significantly higher activity than the original 1.6 kb fragment. Nucleotide sequence comparison of the promoter region of human collagenase-3 revealed a stronger similarity to the mouse collagenase-1 promoter than to the human collagenase-1 promoter.

Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.

Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.

Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.

Repetitive Alu elements form a cruciform structure that regulates the function of the human CD8 alpha T cell-specific enhancer.

We previously identified a T cell-specific enhancer in the last intron of the human CD8 alpha gene that is adjacent to a sequence element that significantly represses enhancer function. This negative regulatory region consists of a half-Alu sequence that has potential to base-pair with a downstream Alu element, which is part of the fully active enhancer, to form a cruciform structure. The activity of this half-Alu silencer sequence is position and orientation-dependent, suggesting that DNA structure plays an important role in its function. Using site-directed mutational analysis and P1 nuclease mapping, we directly demonstrate that formation of a cruciform structure is required for repression of enhancer function in transient transfection assays. Finally, a P1 nuclease-sensitive site is present in the endogenous CD8 alpha gene in T cell lines providing indirect evidence that the stem-loop may form in vivo. Taken together, these results suggest that Alu elements may contribute to the regulation of the CD8 alpha gene enhancer through the formation of secondary structure that disrupts enhancer function.

Identification and characterization of an Alu-containing, T-cell-specific enhancer located in the last intron of the human CD8 alpha gene.

Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.

CD8 functions as an inhibitory ligand in mediating the immunoregulatory activity of CD8+ cells.

Antisense and sense transfection technologies were employed in cellular coculture systems to investigate the physiologic requirements for CD8-dependent immunoregulation. Our data indicate that cells bearing genetically engineered CD8 molecules incorporating a glycoinositolphospholipid membrane anchor, as well as fixed cells bearing natural CD8 molecules, retain specific, CD8-dependent immunoregulatory activity. These findings together support the novel concept that CD8, a molecule traditionally thought of as a receptor, can function as an inhibitory ligand. CD8-dependent inhibition was shown to induce nonresponsiveness, persisting for at least 24 h, in Ag-specific responders. Moreover, only cells undergoing primary, but not secondary, antigenic stimulation through their TCR were found to be susceptible to CD8-dependent inhibition. Both CD4+ and CD8+ responding T cells were inhibited by CD8+ modulatory cells. These functional analyses of inhibitory and responder cells in CD8-dependent inhibition lay the groundwork for developing artificial CD8-based immunomodulatory peptides and deciphering CD8's role in natural immunoregulation.

Regulation of allogeneic responses by expression of CD8 alpha chain on stimulator cells.

By antisense-mediated inhibition of CD8 expression in T cell clones and expression of CD8 in non-T cell lines, we have produced several sets of CD8+/CD8- paired cell lines. These cellular reagents have allowed us to assess the effect of CD8 surface expression on the immunogenic potential of stimulator cells. We found that the presence of CD8 on stimulator cells markedly decreased their capacity to stimulate proliferative responses or to induce the generation of cytotoxic activity. The CD8 alpha chain, in the absence of the beta chain, was sufficient to mediate this inhibitory effect. Our findings support the notion that CD8 functions as an immunoregulatory ligand and that auxiliary cell surface molecules on stimulator cells can have profound effects on the immunogenic capabilities of these cells.

An immunoregulatory function for the CD8 molecule.

The molecular details of immunoregulatory phenomena associated with CD8+ T lymphocytes have not been clearly elucidated. We tested the hypothesis that the cell surface glycoprotein CD8 is itself essential in mediating the inhibitory effects associated with CD8+ T cells. For this purpose we utilized a T-cell clonal pair, consisting of a human CD8+ T-cell clone and a specific CD8- phenocopy of this clone obtained via antisense RNA mutagenesis, to modulate allogeneic responses in vitro. Our findings indicate that the expression of the CD8 molecule by the inhibitory cells is essential for down-regulation of both allogeneic proliferation and generation of cytotoxicity in mixed lymphocyte cultures. These results define an immunomodulatory function for the CD8 molecule and provide insights into the molecular basis of immunosuppression.

Functional consequences of anti-sense RNA-mediated inhibition of CD8 surface expression in a human T cell clone.

An experimental approach for defining the function of CD8 has been developed by linking anti-sense RNA mutagenesis and T cell cloning technologies. We have transfected an anti-sense CD8 episomal expression vector into a CD8+ nontransformed human T cell clone that is specific for the human class I alloantigen HLA-B35. Expression of CD8 on this T cell clone, JH.ARL.1, was selectively and efficiently inhibited. Stimulation of this CD8- variant with specific alloantigen resulted in a marked loss of a number of functional responses, including cytotoxicity, proliferation, IL-2 secretion, and IL-2-R expression. However, these same functional responses could be elicited with stimuli that do not require antigen recognition to activate the T cell (anti-CD3 mAbs, PHA). The results of our study support the hypothesis that CD8 is required for recognition of class I MHC alloantigens that results in activation of T cell functional responses.

Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone.

A methodology was developed for stable gene transfer into cloned nontransformed human T lymphocytes. Stable high-level gene expression was achieved in cloned human T cells by using a self-replicating Epstein-Barr virus (EBV) episomal replicon. A comparison of five eukaryotic promoters established that the Rous sarcoma virus 3' long terminal repeat (RSV 3' LTR) and the lymphopapilloma virus (LPV) 5' LTR are optimal for episome-based expression in T cells. Effective (greater than 95%), selective, and reversible anti-sense RNA-mediated gene inhibition of a model T-cell-associated molecule (CD8) was achieved in a cytotoxic human T-cell clone by using an EBV episome-based, RSV 3' LTR-driven expression system. The linking of anti-sense RNA mutagenesis and T-cell cloning technologies should contribute significantly to studies of human T-cell function.

Kinetics of synthesis and phosphorylation of respiratory syncytial virus polypeptides.

The kinetics of synthesis of [35S]methionine-labelled respiratory syncytial virus-specific proteins were studied in CV-1 cells infected at high multiplicity. Immunoprecipitated viral proteins resolved by SDS-PAGE were quantified by scanning fluorographs of protein bands. The nucleocapsid (N) protein was detectable by 2 h post-infection (p.i.), whereas the phospho- (P), matrix (M) and fusion (Fo) proteins and Vp24 (a matrix-like protein) were first detected between 4 and 6 h p.i. Synthesis of the glyco- (G) protein was first detected at 6 h p.i. and reached its peak synthesis rate at 10 h p.i. Virus-specific P, M and Vp24 proteins were phosphorylated in infected cells. The P protein was highly phosphorylated in purified virions whereas phosphorylated species of the M and Vp24 proteins were minor components. The phosphorylated form of the P protein was detected by monoclonal antibody precipitation, confirming the identity of this protein. The N protein was not phosphorylated in infected cells or in virions. Synthesis of [35S]methionine-labelled proteins preceded detectable 32Pi labelling by several hours. The putative phosphorylated M protein was detected at 6 h p.i. before phosphorylated forms of P and Vp24 were seen. The timing of appearance of the phosphorylated species of P and Vp24 proteins in infected cells corresponded to the release of infectious virions from infected cell monolayers at 10 to 12 h p.i.

Economic policy, intergenerational equity, and the Social Security Trust Fund buildup.

For the next 75 years, the Old-Age, Survivors, and Disability Insurance (OASDI) system is projected to be close to in balance, on average. For approximately the next 40 years, under current projections, the combined OASDI Trust Fund is expected to continually have excesses of income over outgo, creating a buildup that will peak in 2030 at about +12 1/2 trillion (roughly 23 percent of the gross national product). Thereafter, the system is projected to be in annual deficit continually until the trust fund is exhausted in 2051. This article focuses on two fundamental issues that must be understood if the potential economic consequences of this buildup are to be evaluated properly. The first issue deals with the fact that the nature of Federal economic policy during the buildup period will determine the ultimate economic impact of the buildup. The second issue concerns the effect of the buildup, and its disposition, on the Social Security program's treatment of one generation of workers compared with another. If a fund is actually accumulated as projected, part of the retirement benefits of the "baby-boom" generation will, in effect, be self-financed. If, however, that fund is used for other purposes--directly or indirectly--future cohorts of workers will be required to fully finance benefits promised to the baby-boom retirees.