The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28.

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.

The NF-kappa B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes.

We explored the role of the NF-kappa B pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-kappaBalpha (inhibitor of kappa B alpha factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-kappa B site around -840 decreased, while ectopic expression of I-kappa B kinase-beta (IKK beta) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKK beta, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-kappa B factors that bind to the NF-kappa B binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-kappa B activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-alpha. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKK beta and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.

Response differences between human CD4(+) and CD8(+) T-cells during CD28 costimulation: implications for immune cell-based therapies and studies related to the expansion of double-positive T-cells during aging.

Since CD28 costimulation is critical for T-cell activation, there is great interest in CD28 as a target for immuntherapeutic approaches. We show that stimulation of human CD4(+) and CD8(+) T-cells differs in their responsiveness to stimulation with anti-CD3/CD28-coated beads, as surrogate antigen-presenting cells. While the CD4(+) subset responded with sustained proliferation, CD8(+) T-cells grew for a limited period only and failed to produce IL-2 beyond the first few days in culture. This decrease is accompanied with an increased rate of apoptosis in CD8(+) T-cells despite Bcl-x(L) expression. The CD8(+) but not the CD4(+) subset developed a reversible double-positive phenotype during CD28 costimulation. This finding may have some bearing on the appearance of double-positive T-cells in human peripheral blood. This double-positive subset was shown to undergo a statistically significantly increase during aging in humans. Taken together, the above data have important implications for immunotherapy and immune senescence.

Primary human CD4+ T cells contain heterogeneous I kappa B kinase complexes: role in activation of the IL-2 promoter.

NF-kappa B transcription factors play an important role in the activation of the IL-2 gene in response to TCR ligation. The release of NF-kappa B factors to the nucleus requires phosphorylation and degradation of the inhibitory kappa-B proteins (I kappa Bs). I kappa B alpha and I kappa B beta phosphorylation is dependent on dual signaling by the TCR and the CD28 accessory receptor. This pathway involves a multisubunit I kappa B kinase (IKK) complex, which includes the IKK alpha (IKK-1) and IKK beta (IKK-2) kinases. We demonstrate that stimulation of primary human CD4+ T cells by CD3/CD28 activates two distinct endogenous IKK complexes, a heterodimeric IKK alpha/beta and a homodimeric IKK beta complex. IKK beta overexpression in a Jurkat cell line resulted in the formation of a constitutively active IKK complex, which was CD3/CD28 inducible. In contrast, ectopic expression of IKK alpha assembled into a complex with negligible I kappa B kinase activity. Moreover, IKK beta, but not IKK alpha, overexpression enhanced transcriptional activation of the CD28 response element in the IL-2 promoter. Conversely, only kinase-inactive IKK beta interfered in the activation of the IL-2 promoter. Sodium salicylate, an inhibitor of IKK beta, but not IKK alpha, activity, inhibited IL-2 promoter activation as well as IL-2 secretion and interfered in activation of both the heterodimeric as well as the homodimeric IKK complexes in primary CD4+ T cells. Taken together, these data demonstrate the presence of an IKK beta-mediated signaling pathway that is activated by TCR and CD28 coligation and regulates IL-2 promoter activity.

Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis.

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin beta receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the "death domain" of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-kappaB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-kappaB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.

Involvement of dsRNA virus in the protein composition and growth kinetics of host Trichomonas vaginalis.

Trichomonas vaginalis harbors a double-stranded (ds)-RNA virus, and the presence of virus is related to upregulated expression and phenotypic variation of a prominent immunogen (Khoshnan A, Alderete JF (1994) J Virol 68: 4035-4038). To further test the influence of virus on T. vaginalis, virus-infected (V+) isolates were compared to virus-free (V-), agar-cloned progeny trichomonads derived from the parental isolates for accumulation of total proteins and cysteine proteinases. Comparative high resolution two dimensional (2D)-SDS-PAGE was performed of trichomonads grown in a chemostat under identical conditions. At least 47 proteins were identified as specifically expressed by representative V+ isolate 347, and approximately 41 spots were specific to the corresponding V- progeny, showing an association between virus and the presence and absence of parasite proteins. Qualitatively and quantitatively dissimilar cysteine proteinase patterns were detected from numerous V+ isolates and the V- progeny. A 2D analysis for isolate 347 showed the appearance of unique proteinase activities for parental parasites and presence of at least one proteinase in the V- progeny. Finally, the V+ T. vaginalis isolate 347, but not the V- isolate 347 progeny nor other V+ isolates, underwent fluctuations in density during chemostat growth allowing for purification of virus particles from the V+ isolate 347 supernatants during decreased parasite density.

Characterization of double-stranded RNA satellites associated with the Trichomonas vaginalis virus.

Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.

Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.

Most Trichomonas vaginalis isolates are carriers of the multisegmented double-stranded RNA (dsRNA) virus. In vitro polymerase assays were performed to demonstrate the RNA-dependent RNA polymerase (RDRP) activity of purified particles. Transcripts which comigrated with the dsRNAs of the virus were readily detected as synthesized products, indicating viral RDRP activity. In addition, smaller-sized dsRNA species, possibly two of approximately 700 bp (s1) and one of 500 bp (s2), were synthesized by purified virus particles of the CsCl gradient surrounding the virus peak. No cross-hybridization with either s1 or s2 and the dsRNA segments occurred, suggesting that s1 and s2 were synthesized from different templates. An RNase A protection assay revealed that the synthesized s1 and s2 polymerase products were double stranded. Furthermore, hybridization of products with strand-specific RNA of s1 generated from cDNA indicated that only one strand was synthesized in vitro. s1 and s2 were not visualized in ethidium bromide-stained agarose gels of dsRNA of infected trichomonads grown in batch cultures. However, dsRNA profiles of the same infected organisms cultivated under defined continuous-flow conditions contained readily detectable levels of s1 and s2, indicating that amplification of s1 and s2 occurred under specific environmental conditions. These newly discovered dsRNAs were not detected in all of the virus-carrying isolates. Finally, it is noteworthy that the s1 and s2 dsRNAs and the RDRP activity were not detected in trichomonal isolates without virus or in virus-negative progeny derived from virus-positive parental isolates. These data indicate the possibility of variations in the number of dsRNAs and/or of the presence of satellites in trichomonads infected with the multisegmented virus.

Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA.

Some isolates of Trichomonas vaginalis carry a double-stranded RNA virus (TVV) and undergo phenotypic variation between surface expression and cytoplasmic expression of a prominent immunogen (P270). Only trichomonads with TVV express P270 on the surface. Northern (RNA) blots using a specific cDNA encoding the repeat element of the phenotypically varying P270 immunogen as a probe showed accumulation of P270 transcript only in isolates with TVV (V+) in contrast to isolates without the virus (V-). To test further the influence of virus infection on P270 mRNA expression, V- progeny, derived from the parental V+ isolates, were tested. Trichomonads of V- progeny, like the fresh clinical V- isolates, also showed no accumulation of P270 mRNA. By immunoblotting with a monoclonal antibody to the key repeated epitope of P270, it was found that V- organisms had quantitatively less immunoreactive protein than the parental V+ isolates. Although V+ and V- isolates contained proteins immunoreactive with the monoclonal antibody to P270, it was necessary to test for the presence of the P270 gene among all the isolates. As expected, Southern blots demonstrated that V+ and V- trichomonads possessed the gene encoding P270. These data suggest that the double-stranded RNA virus of T. vaginalis is involved in the regulation of P270 mRNA accumulation.

Multiple double-stranded RNA segments are associated with virus particles infecting Trichomonas vaginalis.

Previous studies demonstrated that some isolates of the sexually transmitted protozoan Trichomonas vaginalis are infected with a nonsegmented, double-stranded RNA (dsRNA) virus. A reexamination of the total dsRNA extracted from several virus-harboring isolates indicated the presence of at least three dsRNAs with sizes ranging from 4.8 to 4.3 kbp. The double-stranded nature of each of the three segments was determined by hybridization experiments using riboprobes of opposite polarities obtained from cDNA generated to each of the segments. All three segments were present in agar clones originating from single organisms of T. vaginalis isolates, suggesting that the three segments were not the result of a mixed population of trichomonads harboring different sizes of dsRNA. The three segments were associated with CsCl-purified virus particles, as evidenced by electron microscopy, and RNAse treatment of the preparation containing virus particles did not destroy the dsRNAs. Finally, the individual dsRNA segments were purified for use as probes to determine whether the three dsRNAs shared any sequence homology. Each end-labeled dsRNA segment did not cross-hybridize to any of the other two segments, a finding consistent with the hybridization of labeled cDNAs to only the segments from which they were derived. These results show that the coding capacity of the dsRNA virus may be at least three times greater than that estimated earlier and illustrates further the complexity of this virus-parasite interrelationship.