The tuberous sclerosis 2 gene product can localize to nuclei in a phosphorylation-dependent manner.

The tuberous sclerosis 2 (TSC2) gene has been genetically mapped to a disease characterized by abnormal cell proliferation that results in the production of tumorous lesions in a variety of tissues. The molecular mechanism for TSC2 mediation of tuberous sclerosis is unclear but it appears to be related to its ability to cytoplasmically interact with a second gene, TSC1, mapping to the disease. These proteins are linked to constraints on cell cycle signaling pathways and therefore envisioned to function as tumor suppressor genes. In previous studies we have demonstrated TSC2 associations with steroid receptor family members and modulation of their gene expression capabilities. Here we provide evidence for TSC2 translocation to the nucleus and a possible role for phosphorylation in both TSC2 translocation and TSC2 modulation of steroid receptor-mediated transcription.

Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities.

Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis-acting sequences of DNA, called hormone response elements (HREs). The identification of novel HREs is fundamental to understanding the physiological function of nuclear receptor-mediated signalling pathways. A number of these receptors are transcriptionally active, or can be induced to an active state, when expressed in the yeast strain Saccharomyces cerevisiae. This aspect of nuclear receptor activity was used to screen random rat genomic DNA fragments for their ability to function as a HRE for the farnesoid X-activated receptor (FXR). An isolated genomic fragment mediated FXR transcriptional activation without the co-expression of the retinoid-X receptor (RXR), a receptor previously thought to be an obligate heterodimer partner for FXR function. This genomic sequence of DNA contained a pair of highly conserved HRE half-sites arranged in an everted orientation and separated by 3 bp (ER3). Furthermore, it was located 240 bp from a highly conserved TATA box motif. A minimal ER3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR-expressing yeast extracts. Using RT-PCR, an expressed mRNA fragment was identified within an 8 kb region downstream of the putative TATA box motif. This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst. These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this strategy might be universally applied to any transcription system that can be reconstituted in yeast.

Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin.

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma isoforms in CV-1 cells co-transfected with rat PPAR alpha and gamma, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA beta-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid beta-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARalpha activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(-)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARgamma, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(-)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(-)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(-)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARalpha and/or PPARgamma isoforms.

Catabolites of cholesterol synthesis pathways and forskolin as activators of the farnesoid X-activated nuclear receptor.

The nuclear receptors are a family of transcriptional mediators that, upon activation, bind DNA and regulate gene transcription. Among these receptors, the farnesoid X-activated receptor (FXR) has recently been identified as one activated by bile acids and farnesol. To investigate the potential of other sterols to activate FXR, as well as to examine relevant relationships among identified activators of FXR, the current study used a mammalian cell transcription assay to quantify and compare activation potential. In addition to the classical bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), FXR was shown to be transcriptionally active in the presence of the androgen catabolites 5alpha-androstan-3alpha-ol-17-one (androsterone) and 5beta-androstan-3alpha-ol-17-one (etiocholanolone), as well as the sterol bronchodilatory drug forskolin. Conversely, cholesterol and several other key precursors to the androgens and bile acids were either not active or only slightly active. Furthermore, it was observed that the bile acid ursodeoxycholate (UDCA) could inhibit DCA and CDCA activation of FXR in a manner parallel to its ability to antagonize DCA and CDCA induction of apoptosis. By far, the most efficacious activator of FXR was forskolin. Interestingly, although it is classically viewed as an initiator of the adenylate cyclase/protein kinase A (PKA) pathway, PKA inhibition did not inhibit forskolin's activation of FXR nor was cyclic AMP (cAMP) able to stimulate FXR-mediated transcription. These data would suggest that forskolin acts as a ligand for FXR rather than as a secondary activator of FXR and could have important implications with respect to its potential toxicity and pharmacological use.

Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members.

Tuberous sclerosis (TSC) is a genetic disorder that results in the development of hamartomatous lesions in a variety of organ systems. Both the prevalence of the disease and the often devastating consequences of these tumors pose a serious health and medical care problem. The disease has been mapped to two distinct genetic loci in humans, and although the genes (TSC1 and TSC2) for both loci have recently been cloned, their function remains an enigma. Data presented here demonstrates that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes. These data place TSC2 into a growing list of nuclear receptor coregulators and strengthen the expanding body of evidence that these coregulators may play critical roles in cellular differentiation.

Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids.

1. The effects of endogenous fatty acids and perfluorooctanoic acid (PFOA) and its analogs on peroxisomal acyl CoA oxidase (ACO) and microsomal laurate hydroxylase (LH) activities were evaluated in primary cultures of rat hepatocytes and activation of peroxisome proliferator-activated receptor alpha (PPARalpha) in CV-1 cells. The rank order for the stimulation of ACO activity in hepatocytes for selected compounds was PFOA >> octanoic acid>octanedioic acid, perfluorooctanol (inactive). Increases in ACO activity by PFOA, like those of ciprofibrate, were associated with a marked increase in peroxisome number and cytosolic occupancy volume. Maximal effects of ciprofibrate and PFOA on the stimulation of ACO activity were not additive, suggesting that these two compounds share a common pathway of peroxisome proliferation. 2. Saturated monocarboxylic acids of C4 to C18 chain length were inactive, and, among dicarboxylic acids, only small elevations (40-45%) in ACO activity were observed with the long-chain C12 and C16 dioic acids. Of the C18 fatty acids tested, only oleic and linoleic acids, at 1 mM, produced a two- to three-fold elevation in ACO and LH activities. In comparison with endogenous fatty acids, PFOA was more potent and exhibited a different time course and greater magnitude of stimulation of ACO and LH activities in cultured hepatocytes. 3. Addition of mitochondrial beta-oxidation inhibitors (3-mercaptopropionic and 2-bromooctanoic acids) did not alter ACO activity in the presence of octanoic acid or octanedioic acid; nor did they modify the stimulation of ACO activity by PFOA. The carnitine palmitoyltransferase I inhibitor 2-bromopalmitic acid produced a 2.5-fold increase in ACO stimulatory activity and reduced both ciprofibrate- and PFOA-mediated stimulations of ACO activity. 4. Cycloheximide treatment reduced PFOA- and ciprofibrate-induced ACO activities; however, the response to oleic acid was not blocked and increased slightly. 5. In rat and human PPARalpha transactivation assays, the rank order of activation was ciprofibrate > PFOA > oleic acid > or = octanoic acid > octanedioic acid or perfluorooctanol (inactive). PFOA, ciprofibrate and oleic acid were activators of rPPARalpha at concentrations that correlated favorably with the changes in ACO activity in cell culture. Octanoic acid did not increase ACO activity and was a weak activator of PPARalpha. 6. Our findings suggest that fatty acids such as oleic acid (endogenous fatty acids) and PFOA (a stable fatty acid) act through more than one pathway to increase ACO activity in rat hepatocytes. We conclude that the potent effects of PFOA are primarily mediated by a mechanism that includes the activation of liver PPARalpha.

Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines.

Epstein-Barr virus (EBV) transformed human B cells proliferate indefinitely in vitro, and it has been proposed that cytokine-mediated autocrine loops contribute to the maintenance of the lymphoblastoid phenotype. We used a novel multiprobe RNase protection assay to quantify cytokine mRNA species expressed by EBV-transformed lymphoblastoid cell lines (LCL), derived either by the transformation of B cells with B95-8 or wild-type EBV or by the in vitro outgrowth of EBV-associated B cell lymphomas to identify cytokines that are commonly expressed in all LCL and thus more likely to be essential for immortalization of B cells. All 16 LCL expressed high levels of tumor necrosis factor (TNF)alpha, TNFbeta, and transforming growth factor (TGF)beta1 mRNA, while interleukin (IL)-10 transcripts were detected in most LCL but at a lower level. Expression of IL-1alpha, IL-1beta, IL-6, IL-12p35, IL-12p40, IL-13 and IFNgamma mRNA was variable among the LCL tested. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, and IL-5 mRNA were undetectable in all LCL. Furthermore, we found that IL-10, TNFalpha, and TNFbeta mRNA were induced in EBV-negative B cell lines after infection with EBV. These data define common versus idiosyncratic patterns of cytokine expression by LCL and, in the former case, such cytokines as TNFalpha, TNFbeta, and IL-10 emerge as strong candidates that are essential for the autocrine regulation of EBV-immortalized B cells.

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor alpha (rPPAR alpha) activation and peroxisomal fatty acid beta-oxidation.

Enantiomers of a series of substituted analogs of 2-(4-chlorophenoxy) -acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the nu-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer >> (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4'-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) >> (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity (R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPAR alpha and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-nu-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site.

Convergence of three steroid receptor pathways in the mediation of nongenotoxic hepatocarcinogenesis.

The mechanisms by which peroxisome proliferators are able to regulate metabolic processes such as fat metabolism, while at the same time creating an environment for the development of hepatocellular carcinomas, is a central issue in the non-genotoxic carcinogenesis field. The convergence of two members of the steroid receptor family (peroxisome proliferator-activated receptor, PPAR; and retinoid X receptor, RXR) has provided strong support for an oxidative stress component in this carcinogenesis process, but has yet to define clearly a pathway for the classical tumor promotion events associated with peroxisome proliferation. The findings presented here integrate a third member of the steroid receptor family into this process and suggest a novel autocrine loop and mechanism for creating both oxidative stress and tumor promotion. A central regulatory component in this pathway is farnesol which has recently been shown to induce transcription mediated by the steroid receptor family member, farnesoid X receptor (FXR). In this report, it is clearly demonstrated that farnesol can also upregulate the transcriptional events of PPAR, but most likely through a different farnesoid metabolite, resulting in the regulation of an entirely different set of genetic components. Deregulation of the activities of these receptors offers a provocative mechanism for explaining the hepatocarcinogenic effects of peroxisome proliferators in chronically treated rodents.

Peroxisome proliferator-activated receptor response specificities as defined in yeast and mammalian cell transcription assays.

Peroxisome proliferators include a heterogeneous group of xenobiotic agents capable of inducing peroxisome proliferation and hepatocellular carcinomas in rodent model systems. These chemicals appear to mediate their activity through a family of transcription factors known as peroxisome proliferator-activated receptors (PPAR). Recently it has been shown that DNA binding of PPAR is contingent upon heterodimerization with a member of the retinoic acid X (RXR) family of receptors. In this report transcription parameters of a rat PPAR alpha were analyzed using mammalian and yeast cotransfection assays. PPAR activity was observed to be peroxisome proliferator dependent in the mammalian cotransfection assay, and heterodimer dependent but peroxisome proliferator independent in a yeast version of the same assay. Moreover, when the naturally occurring ligand for RXR, 9-cis-retinoic acid (RA), was tested in the same assays, it was observed to generate an RXR-specific response in the yeast cell assay but host cell-specific response in the mammalian cell assay. Finally, the combination of peroxisome proliferator and 9-cis-RA had very little added effect on the yeast cell assay but again produced a cell-specific synergistic response in the mammalian cell assay. These data demonstrate that PPAR transcriptional activity is strongly influenced by the RXR family of receptors, and that peroxisome proliferators may be regulating PPAR mammalian cell activity through a secondary mechanism.

Identification of a nuclear receptor that is activated by farnesol metabolites.

Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represent targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.

The mouse model system was used to evaluate age-associated changes in the subset composition and function of the splenic CD8+ T cell pool. In response to stimulation with plate-bound anti-CD3 epsilon mAb, CD8+ cells from old C57BL/6NNia mice produced greater levels of IFN-gamma than cells from young-adult controls. This age-associated difference was apparent at the levels of both IFN-gamma mRNA accumulation and cytokine release, and was established within the first major cell cycle in culture. In addition, the capacity to produce IFN-gamma appeared to increase gradually with age as evidenced by studies on CD8+ cells from intermediate aged mice. In contrast to these findings, the peak S-phase responses by stimulated CD8+ cells from old mice were significantly reduced relative to young-adult controls. Immunophenotypic analyses of membrane CD44, CD45RB, 3G11, and MEL-14 expression by splenic CD8+ cells from young-adult, intermediate-aged, and old mice revealed an age-associated decrease in the frequencies of cells that expressed low levels of CD44 and high levels of CD45RB, 3G11, and MEL-14, whereas the reciprocal phenotypes increased with age. The correlated analysis of all four subset markers identified a composite phenotype (CD44loCD45RBhiMEL-14hi3G11lo/hi) which, based on past functional studies, is a candidate phenotype for naive cells. This "naive" phenotype dominated the CD8+ cell pool of young-adult mice but decreased in frequency with age. In contrast to the CD44lo cell group, the CD44hi cell fraction, which is associated with preactivated/memory CD8+ cells, was found to be uniformly 3G11lo but expressed heterogeneous levels of CD45RB and MEL-14, perhaps defining multiple subsets within the memory population. All of these latter subsets increased in frequency with age. Finally, we found that when CD8+ cells were fractionated based on CD44 expression the capacity to release measurable levels of IFN-gamma segregated entirely with the CD44hi fraction, irrespective of donor age. Together, these findings support the hypothesis that aging is accompanied by dramatic shifts in the subset compositions of splenic CD8+ cell pools, which contribute significantly to their increased capacity to produce IFN-gamma at the population level.

Patterns of cytokine gene expression by CD4+ T cells from young and old mice.

We have analyzed the patterns of induced cytokine gene expression and cell cycle activity by CD4+ cells from mice, and have examined how these response patterns change during the aging process. CD4+ cells were isolated from spleens of young adult and old C57BL/6NNia mice and were stimulated in vitro with plate-bound anti-CD3 epsilon mAb. The cells were then assessed over time for the capacity to accumulate transcripts for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta; to secrete IL-2, IL-3, IL-4, IL-5, IL-6, and IFN-gamma; and to progress through S phase. Before the first major cell division in culture (< 32 h), stimulated CD4+ cells of the old group contained similar peak levels of IL-2, TNF-alpha, and TNF-beta transcripts relative to young adult controls, whereas IL-3, IL-4, IL-5, and IFN-gamma transcripts accumulated to significantly higher peak levels in the old group. These findings were consistent with the patterns of cytokine secretion later in culture (24 to 72 h): the peak IL-2 levels were similar between age groups, but the old group exhibited an enhanced capacity to release IL-3, IL-4, IL-5, and IFN-gamma. In contrast, CD4+ cells of the young group were superior in the hyper-expression of the housekeeping gene, rpL32, before cell division and in the levels of S phase activity throughout 3-day cultures. Similar analyses of CD4+ cells from mice of intermediate ages showed that the alterations in cytokine profiles occurred gradually from young adulthood to old age, whereas the reductions in proliferative capacity were late life changes. Consistent with previous reports, we found that the splenic CD4+ cell group also underwent a progressive, age-dependent increase in the proportions of cells expressing high levels of membrane CD44 (a phenotype associated with memory or effector cells). Moreover, the analysis of IL-3, IL-5, and IFN-gamma production by isolated CD4+CD44lo and CD4+CD44hi cells revealed that the capacity to produce these cytokines segregated predominantly with the CD44hi subset, regardless of donor age. Taken together, our data suggest that gradual age-associated shifts in the subset composition of the splenic CD4+ cell pool underlie progressive changes in the patterns of cytokine gene expression by this cell group.

Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.

Peroxisomes are cytoplasmic organelles which are important in mammals in modulation of lipid homeostasis, including the metabolism of long-chain fatty acids and conversion of cholesterol to bile salts (reviewed in refs 1 and 2). Amphipathic carboxylates such as clofibric acid have been used in man as hypolipidaemic agents and in rodents they stimulate the proliferation of peroxisomes. These agents, termed peroxisome proliferators, and all-trans retinoic acid activate genes involved in peroxisomal-mediated beta-oxidation of fatty acids. Here we show that the receptor activated by peroxisome proliferators and the retinoid X receptor-alpha (ref. 6) form a heterodimer that activates acyl-CoA oxidase gene expression in response to either clofibric acid or the retinoid X receptor-alpha ligand, 9-cis retinoic acid, an all-trans retinoic acid metabolite; simultaneous exposure to both activators results in a synergistic induction of gene expression. These data demonstrate the coupling of the peroxisome proliferator and retinoid signalling pathways and provide evidence for a physiological role for 9-cis retinoic acid in modulating lipid metabolism.

Identification and characterization of the effector region within human C5a responsible for stimulation of IL-6 synthesis.

Human C fragment C5a is known to be a proinflammatory mediator and more recently shown to be a potent modulator of both humoral and cell-mediated immunity. We recently reported that natural and recombinant C5a induces the synthesis of IL-6-specific mRNA and secreted protein from human monocytes. Our studies using analogue peptides that are homologous to the carboxyl-terminal sequence of human C5a, indicate that the "effector" site for inducing IL-6 synthesis resides within the C-terminal region (C5a (70-74)) of the C5a molecule. C5a peptides containing the exact sequence of the natural factor were found to retain full agonist activity but exhibited low potency (0.01-0.1% of intact C5a). It was also shown that amino acid substitutions in the C5a peptides by aromatic/hydrophobic residues, outside the immediate effector site, resulted in analogue peptides with a substantial increase in potency relative to the most active natural peptide (C5a (56-74)). Moreover, these peptides approach the potency of natural C5a for induction of IL-6. Taken together, these results suggest that the inflammatory and immunoregulatory activities associated with C5a may, in part, be due to the synthesis of IL-6.

Cell proliferation and cytokine production by CD4+ cells from old mice.

Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3 epsilon mAb, 145-2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at approximately 48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were approximately 40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell-depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were approximately 10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFN gamma mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFN gamma transcripts, than young donor CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 production by murine B cells and B cell lines.

We have analyzed interleukin (IL)-6 gene transcription and IL-6 secretion by murine B cells in vitro. Mitogenic doses of lipopolysaccharide (LPS) or LPS in combination with F(ab')2 goat anti-mouse IgM antibodies (GAMmu), but not GAMmu alone, induced B cells to synthesize and release IL-6. In time course experiments, the accumulation of IL-6 mRNA was first detectable at 24-36 hr of culture and the levels were maintained through 60 hr; these kinetics correlated well with increases in supernatant IL-6 levels and were coincident with vigorous cell cycle activity. We also analyzed constitutive and LPS-induced IL-6 gene expression by the murine B cell lines: 70Z/3, 38C-13, WEHI-231, X16C, WEHI-279, and BCL1. Only the WEHI-279 and BCL1 lines produced detectable IL-6 constitutively, and the BCL1 cells could be further induced by treatment with LPS. Of the remaining cell lines, only WEHI-231 and X16C could be stimulated with LPS to produce IL-6. To evaluate whether IL-6 could influence proliferation and Ig secretion by the cell lines, low cell density cultures were established in the presence of various doses of human rIL-6 and were assessed over time for levels of [3H]thymidine uptake and supernatant Ig. Under these conditions, IL-6 had no effect on either cell function.