Induction of granulysin in CD8+ T cells by IL-21 and IL-15 is suppressed by human immunodeficiency virus-1.

Immunosuppression following infection with HIV-1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb. Granulysin, expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV-1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human CD8+ T cells, in this study, we identify IL-21 as a strong inducer of granulysin, demonstrate that IL-21 and IL-15 activate granulysin expression within CD8+ CD45RO+ T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within CD8+ T cells. We show that infection of PBMC from healthy donors in vitro with HIV-1 suppresses granulysin expression by CD8+ T cells, concomitant with reduced p-STAT3 and p-STAT5, following activation with IL-15 and IL-21. Of note, simultaneous signaling through IL-15 and IL-21 could partially overcome the immunosuppressive effects of HIV-1 on granulysin expression by CD8+ T cells. These results suggest that HIV-1 infection of PBMC may reduce the antimicrobial profile of activated CD8+ T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV-1 on CD8+ T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV+ patients and for informed guidance of cytokine-based therapy to restore T cell function.

Alterations in NF-kappaB and RBP-Jkappa by arenavirus infection of macrophages in vitro and in vivo.

Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-kappaB and RBP-Jkappa, we compared the activities of NF-kappaB and RBP-Jkappa in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-kappaB activation and caused a shift in the size of the RBP-Jkappa complex. The virulent P18 virus showed less inhibition of NF-kappaB and failed to alter the size of the RBP-Jkappa complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-kappaB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jkappa. By contrast, P18 induced large amounts of the NF-kappaB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jkappa size. Taken together, these changes suggest that the attenuated viral strain induces an "activation" of macrophages, while the virulent form of the virus does not.

Alterations in mitochondria and mtTFA in response to LPS-induced differentiation of B-cells.

Stimulation of immune cells results in altered cell function and metabolism, which must be recognized by and coordinated with energy production from mitochondria. Mitochondria contain their own DNA genome encoding 13 polypeptides that combine with nuclear-derived subunits to create functional enzyme complexes of the electron transport chain. Therefore, coordination of mitochondrial and nuclear transcription is necessary to achieve a sustained elevation in mitochondrial ATP production. Pre-B-lymphocytes stimulated with lipopolysaccharide exhibit increased activity levels of the mitochondrial enzymes, succinate dehydrogenase and cytochrome c oxidase. Immunoblot analyses of purified mitochondria indicate an increase in the mitochondrial transcription factor (mtTFA) levels in mitochondria induced by cell stimulation. This increase is consistent with increased mtTFA production in the cytoplasm. In addition, mitochondrial protein extracts indicate an increase in protein binding to a mtTFA-DNA binding site from the mitochondrial genome, subsequent to cell stimulation. These results indicate that mitochondrial activity changes during B-lymphocyte stimulation, and mtTFA may contribute to the coordination of respiration with cellular energy demand.

Aptamers containing thymidine 3'-O-phosphorodithioates: synthesis and binding to nuclear factor-kappaB.

Aptamers targeting NF-kappaB containing thymidine 3'-O-phosphorodithioates in selected positions of an oligonucleotide duplex were synthesized. Binding affinities to NF-kappaB varied with the number and positions of the dithioate backbone substitutions. One of the aptamers showed specific binding to a single NF-kappaB dimer in cell culture extracts.

Kinase-independent potentiation of B cell antigen receptor-mediated signal transduction by the protein tyrosine kinase Src.

Signal transduction mediated by the B cell Ag receptor involves the activation of multiple protein tyrosine kinases that are members of the Src family (i.e., Fyn, Lyn, Blk, Lck). To determine whether members of the Src family possess common physical and/or enzymatic properties that enable them to potentiate signal transduction via the B cell Ag receptor, we expressed the protein tyrosine kinase Src in the B lymphoma cell line K46-17 mu m lambda. Based on coprecipitation analysis and two-color immunofluorescence, this heterologous Src family kinase was observed to physically associate with the B cell Ag receptor. Additional experiments demonstrated that B cell Ag receptor cross-linking results in increased tyrosine phosphorylation and activation of Src. Several parameters of B cell activation, including tyrosine phosphorylation of intracellular substrates, calcium mobilization, and transcription factor activation, were potentiated in cells that expressed Src when compared with control cells. To determine whether potentiation of Ag receptor-mediated signaling by Src was dependent on its catalytic activity, a kinase-deficient form of Src was expressed in K46-17 mu m lambda cells. Transfectants expressing kinase-deficient Src exhibited an enhanced responsiveness to stimulation through the B cell Ag receptor that was comparable with transfectants expressing wild-type Src. Additionally, kinase-deficient Src was observed to associate with the endogenous kinase Lyn in an activation-dependent manner. These findings indicate that members of the Src family may potentiate Ag receptor-mediated signaling via a kinase-independent mechanism(s) that involves amplification of kinase recruitment to the Ag receptor activation complex.

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion.

Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.

Tumor necrosis factor and the pathogenesis of Pichinde virus infection in guinea pigs.

Pichinde virus (PIC) is a reticuloendothelial arenavirus of the New World tropics. A guinea pig passage-adapted strain of this virus (adPIC) is uniformly lethal for inbred guinea pigs, while the related, prototype strain (PIC3739) has attenuated virulence. The abilities of adPIC and PIC3739 to induce tumor necrosis factor (TNF) in vivo and in cultured macrophages were compared. Infection with adPIC, but not PIC3739, was associated with detectable serum TNF that peaked in week 2 of infection. Tumor necrosis factor was found in the spleens of adPIC- and PIC3739-infected animals in week 1 of infection; TNF alpha mRNA levels in spleens and livers of adPIC infected animals increased and remained high throughout infection, whereas PIC3739-infected organs showed down regulation of TNF alpha mRNA late in infection. Peritoneal macrophages explanted from adPIC-infected animals showed enhanced lipopolysaccharide-inducible TNF production. Altered regulation of TNF production may play a role in the pathogenesis of guinea pig arenavirus disease.

Shigella flexneri-HeLa cell interactions: a putative role for host cell protein kinases.

Epithelial cell invasion has been shown to be a prerequisite for Shigella flexneri virulence. Recently, we have documented the induction of transcription factor DNA binding activities as a result of S. flexneri challenge of HeLa cells. In this report, we show that HeLa cells challenged with S. flexneri display differences in phosphotyrosine-containing proteins. These changes are detected as early as 5 min post-challenge. Challenge with a noninvasive ipaB mutant strain resulted in the induction of a similar, but less intense, profile of phosphotyrosine-containing host cell proteins. Phosphotyrosine-containing proteins could be detected in S. flexneri, but were unique from those detected following HeLa cell challenge. S. flexneri invasion of HeLa cell monolayers was reduced by treatment with protein kinase inhibitors. These data suggest a role for protein kinases in the initial response of host cells to S. flexneri.

Modulation of cisplatin cytotoxicity by sulphasalazine.

The efficacy of cisplatin [cis-diamminedichloroplatinum (II); DDP] is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance (IC50 values of 25.0 +/- 3.9 vs 4.5 +/- 1.0 microM respectively). Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.

Gender-related differences in expression of murine glutathione S-transferases and their induction by butylated hydroxyanisole.

The basal levels of mu and pi class glutathione S-transferases RNA were 18-fold higher in the male mouse liver as compared with the female. When 0.75% (w/w) BHA was included in the diet it altered the RNA levels of alpha, mu, pi GST classes and mGSTA4-4 in a tissue and sex specific manner. The most marked induction of RNA was seen for the mu class GSTs of female liver, lung and kidney (52, 10 and 8-fold, respectively), and of male liver and kidney (25 and 3.5-fold, respectively), the pi class GSTs of female liver, lung, and kidney (11, 10, and 5-fold, respectively), and mGSTA4-4 of female liver (4-fold). The effect of BHA on the induction of the mu and pi class GST RNA was 2-9 fold greater in female as compared with male tissues. The degree of induction of GST RNA did not correlate directly with changes in GST protein indicating that post-transcriptional events regulating GST expression may be affected by BHA particularly for GST mu and mGSTA4-4.

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.

Shigella flexneri invasion of HeLa cells induces NF-kappa B DNA-binding activity.

Although information about the genetic basis and mechanisms of Shigella flexneri cellular invasion is accumulating, little is known about changes in cell signaling and their consequences following bacterium-host cell interactions. A general result of signal transduction is alterations in the levels and/or activities of transcription factors. Alterations in transcription factor binding activities were observed following challenge with S. flexneri. Changes in the DNA-binding activities of cellular transcription factors to AP1, AP2, cyclic AMP response element, CTF1/NF1, NF-kappa B/Rel, OCT1, and SP1 DNA-binding sites were investigated by electrophoretic mobility shift assays. NF-kappa B/Rel DNA-binding activity was enhanced more than 11-fold by cellular invasion; noninvasive S. flexneri strains induced low levels of kappa B DNA binding. Both subunits of the NF-kappa B transcription factor, p50 and p65, but not c-Rel (p85), are components of the kappa B DNA-binding activity. These data suggest that changes in cellular transcription factor binding activity are a consequence of S. flexneri invasion, and these changes could play a role in the initial host response or in the pathogenesis of the disease.

The physical interactions between p37env-mos and tubulin structures.

The c-mos protein has been reported to be complexed with tubulin and to co-localize with microtubules in unfertilized Xenopus eggs as well as in NIH3T3 cells transformed by the Xenopus c-mos gene. We performed experiments to determine whether the viral mos protein, p37v-mos, also associates with tubulin. Both mouse c-mos and v-mos proteins synthesized in vitro co-polymerized with tubulin. Upon incubation at 37 degrees C, essentially all of the mos protein (both viral and cellular) co-polymerized with tubulin, while more than 50% of the tubulin remained in the depolymerized state. The mos-tubulin interaction was specific, as indicated by the insolubility of the v-mos protein following a second cycle of temperature-dependent depolymerization/polymerization. Beta-tubulin was shown to co-precipitate with p37v-mos and to be phosphorylated by the mos kinase in vitro. Although both v-mos and c-mos proteins co-polymerize with tubulin, p37v-mos behaved differently from p39c-mos on gel filtration columns under conditions that favor disassembly of microtubules. Like Xenopus c-mos, the bulk of the mouse c-mos protein synthesized in vitro appeared in structures that fractionate at about 500 kDa. In contrast to c-mos, the majority of the v-mos protein, either isolated from stably transformed NIH3T3 cells or synthesized in vitro, eluted in the 100 kDa fraction, co-fractionating with tubulin dimers. Therefore, the v-mos protein appears to have a higher affinity for unpolymerized tubulin than c-mos, under conditions that favor disassembly of microtubules.

v-mos protein produced by in vitro translation has protein kinase activity.

The v-mos protein, termed p37v-mos, has a closely associated serine/threonine protein kinase activity. To provide further information about its protein kinase activity, we tested the activity of p37v-mos produced in a cell-free translation system from transcripts generated from a cloned v-mos gene. Anti-mos(37-55) immunoprecipitates of in vitro-produced p37v-mos were found to possess serine/threonine protein kinase activity, whereas those obtained with anti-mos(260-271), known to block v-mos autophosphorylation, lacked kinase activity. The phosphorylated products were identical in size to p37v-mos and p43v-mos produced in protein kinase assays from Moloney murine sarcoma virus-infected cells expressing authentic p37v-mos. These results provide further proof that the protein kinase activity associated with p37v-mos is an intrinsic property of the v-mos gene product. This translation system also provides a useful experimental model to study the activation of the mos protein kinase. Thus, protein kinase assays performed on [35S]methionine-labeled p37v-mos produced p43v-mos at the expense of p37v-mos. Phosphatase treatment removed the p43v-mos species, resulting in increase of the p37v-mos-sized protein, confirming our previous interpretation that p43v-mos is a hyperphosphorylated form of p37v-mos.

Evidence for somatic cell expression of the c-mos protein [corrected].

The c-mos protein has been found to be enriched in germ cells of male mice, as described in a recent report from this laboratory (Herzog et al., Oncogene 3, 225, 1988). We report on further studies which indicate that the c-mos protein (a 41 to 43 kDa protein termed p43c-mos) is expressed in somatic tissues of mice and in cells grown in culture. In testes of mice, germ cell fractions have increased levels of p43c-mos relative to other cells of the testes. However, non-germ cells harbor significant levels of p43c-mos, as judged by comparison of testes from normal mice to those with mutations that affect the germ cell content of the testes. Thus, homozygous S1, at, and the W/Wv mutant mice are sterile due to severe deficiencies of germ cells. Such mice had only an estimated 50%-60% reduction in p43c-mos as judged by western immunoblotting using two different site-directed anti-mos antibodies. Similarly, X/X-sex reversed mice in which germ cells die after 10 days of age had only an 85% reduction of p43c-mos in mice 35 days of age. Thus, the germ cell content of testes did not correlated with p43c-mos levels in this tissue. Direct analyses of non-germ cells derived from mouse testes confirmed these findings, since Sertoli and Leydig cell lines grown in culture expressed p43c-mos. In addition, tissues such as kidney, liver, spleen and brain were found to contain p43c-mos. Surprisingly, mouse NIH3T3 cells were found to express significant levels of the c-mos protein based upon immunoblotting and one-dimensional peptide mapping experiments performed with both anti-mos antibodies. The concentration of the c-mos protein was not affected by expression of viral mos proteins. We conclude that the c-mos protein is enriched in male germ cells, but p43c-mos is also expressed in significant amounts in somatic tissues and in fibroblastic cells grown in culture.

Identification of the protein product of the c-mos proto-oncogene in mouse testes.

The mouse c-mos proto-oncogene RNA is expressed primarily in mouse gonadal tissues and embryos. Until now, the c-mos protein has not been identified. Utilizing two different site-directed affinity purified anti-peptide antibodies, we have identified a 43 kDa c-mos protein in mouse testes and in germ cell preparations derived from testes. This 43 kDa testicular protein was found to be structurally related to a bacterially expressed c-mos protein by peptide mapping. Immunoblots of whole mouse sections were employed to establish that the c-mos protein is expressed primarily in the testes.

The P55 protein affected by v-mos expression is vimentin.

Rabbit antiserum prepared against a cyclic 19-amino-acid peptide predicted from the sequence of the viral mos gene (v-mos) of Moloney murine sarcoma virus not only recognized v-mos gene products but also specifically detected a 55,000-Mr polypeptide expressed in a variety of cells that grow on solid surfaces. This normal cellular protein, previously shown to be reduced in amount in cells expressing the v-mos gene, was found to be the intermediate filament structural protein, vimentin. This conclusion was reached by comparing relative mobilities in denaturing gels, isoelectric points, immunoreactivities, location in the cell, and peptide maps.