The T cell receptor repertoire of CD4-8+ thymocytes is altered by overexpression of the BCL-2 protooncogene in the thymus.

The bcl-2 gene encodes an intracellular, membrane-associated protein that protects immature cortical thymocytes from a wide variety of apoptotic stimuli, including glucocorticoids, radiation, and anti-CD3 treatment. Since cortical thymocytes are the primary target cells for thymic positive and negative selection processes, and since these processes are associated with cell death, we evaluated the role of bcl-2 in T cell development in two ways. In the first approach, transgenic mice expressing high levels of Bcl-2 in cortical thymocytes were mated with H-Y T cell receptor (TCR) transgenic mice, the latter being a well-defined system for the study of positive and negative selection of T cells. We found that the bcl-2 transgene had a dramatic effect on positive selection. This was manifested by a greatly increased production of mature thymocytes that were highly skewed towards the CD4-8+ lineage. The change involving CD4-8+ thymocytes occurred not only in bcl-2 transgenic mice, but was also observed in H-Y TCR/bcl-2 doubly transgenic mice, regardless of whether the H-Y TCR was expressed in the selecting (H-2b) or nonselecting (H-2d) environments. Furthermore, a large proportion of CD4-8+ thymocytes produced in H-2b H-Y TCR/bcl-2 doubly transgenic female mice expressed endogenous TCR alpha chains rather than the transgenic TCR alpha chain. These observations are consistent with the model that high expression of Bcl-2 in cortical thymocytes overrides the normal apoptotic pathway. This then allows the selection of CD4-8+ thymocytes expressing TCRs that are otherwise nonselectable. However, the bcl-2 transgene did not protect CD4+8+ thymocytes expressing the male-specific TCR from deletion in male doubly transgenic mice. In the second approach, we determined the level of bcl-2 mRNA expression in populations of thymocytes defined by their CD4/CD8 phenotypes using quantitative reversed transcriptase PCR techniques. Our results indicate that bcl-2 mRNA was expressed at a high level in immature CD4-8- thymocytes and in mature CD4+8- thymocytes. There is a dramatic downregulation of bcl-2 mRNA in CD4+8+ thymocytes, particularly those expressing a low level of TCR. CD4+8+ thymocytes that upregulated their TCR, likely as a result of receiving positive selection signals, also upregulated bcl-2 mRNA. This observation suggests that rescue of immature thymocytes from the programmed cell death pathway by positive selection signals is accompanied by the upregulation of bcl-2 mRNA.

B cell stimulating factor 2/interleukin 6 is a costimulant for human thymocytes and T lymphocytes.

Growth and differentiation of thymocytes and mature T lymphocytes is regulated by cellular interactions that are in part mediated by soluble factors. We identify IL-6, formerly called B cell stimulating factor (BSF-2). IFN-beta 2, or hybridoma-plasmacytoma growth factor (HPGF) as a novel T cell costimulant rIL-6 induced a six-to seven-fold increase in proliferation of human thymocytes stimulated with suboptimal doses of PHA. A similar effect with added IL-6 could be observed using peripheral blood T lymphocytes, but only if the cultures were first rigorously depleted of monocytes that release high levels of IL-6. Analysis of the mechanism of the IL-6 effect on thymocytes and T lymphocytes showed that IL-6 did not lead to an increase in IL-2-R expression. Concentrations of antibody to IL-2-R inhibiting IL-2 effects did not block the IL-6-induced proliferation, indicating that the IL-6 effect was relatively IL-2 independent. These results identify IL-6 as a novel costimulant of human thymocytes and mature T lymphocytes, and suggest that IL-6 is also an important regulatory of cellular immunity.

Isolation and characterization of a light chain variable region gene for human rheumatoid factors.

Previously, we isolated a Vk gene (Humkv325) from a human placenta that encodes RF light chains bearing the PSL2 and PSL3 CRI markers. Here we report the isolation and characterization of a second human Vk gene (Humkv328) that can be used for RF synthesis. This Vk gene probably encodes at least two 6B6.6 CRI+ RF light chains (Les and Pom) from unrelated subjects, and thus may be related to the light chain-associated 6B6.6 CRI.

Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice.

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene-expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF-expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self-antigens. This physiologic role of RF B cells may be disrupted in RF-associated autoimmune disease.

Neonatal hepatitis induced by alpha 1-antitrypsin: a transgenic mouse model.

Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of the human alpha 1-antitrypsin (alpha 1-Pi) gene. All of the alpha 1-Pi transgenic mice expressed the human protein in the liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The human M-allele protein was secreted normally into the serum. However, the human Z-allele protein accumulated in several cell types, but particularly in hepatocytes, and was found in serum in tenfold lower concentrations than the M-allele protein. Mice in one lineage carrying the mutant Z allele expressed high levels of human alpha 1-Pi RNA and displayed significant runting (50% of normal weight) in the neonatal period. This lineage was found to have alpha 1-Pi-induced liver pathology in the neonatal period, concomitant with the accumulation of human Z protein in diastase-resistant cytoplasmic globules that could be revealed in the Periodic acid-Schiff reaction (PAS). The phenotype of mice in the strain expressing high levels of the Z allele is remarkably similar to human neonatal hepatitis, and this strain may prove to be a useful animal model for studying this disease.

Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1.

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.

Antitumor drug fostriecin inhibits the mitotic entry checkpoint and protein phosphatases 1 and 2A.

In most eukaryotic cells, entry into mitosis is tightly controlled and requires completely replicated and undamaged DNA. We show that the antitumor drug, fostricin, interferes with this control; it induces cycling cells to enter mitosis prematurely, and it can overcome the mitotic entry checkpoint, forcing into mitosis cells that were arrested in the division cycle by treatment with the DNA replication inhibitor aphidicolin or with the DNA-damaging agents camptothecin and teniposide. This effect was observed in all rodent, simian, and human cell lines tested. Fostriecin also hampers progression through the later stages of mitosis as determined by the absence of normal half-spindles, anaphase figures, and telophase figures. The only previously known target for fostriecin is topoisomerase II, which is inhibited in vitro with a 50% inhibitory concentration of 40 microM (T. J. Boritzki, T. S. Wolfard, J. A. Besserer, R. C. Jackson, and D. W. Fry. Inhibition of type II topoisomerase by fostriecin. Biochem. Pharmacol., 37: 4063-4068, 1988). We show that fostriecin is a more potent inhibitor of protein phosphatase 1, with a 50% inhibitory concentration of 4 microM and protein phosphatase 2A, with a 50% inhibitory concentration of 40 nM. Inhibition of the mitotic entry checkpoint and inhibition of protein phosphatases are novel properties for antitumor drugs with potential or proven therapeutic value.

Mouse retroviral sequences acquired by cell lines after passaging through nude mice detected by hybridization of the fms probe pSM3.

The expression of a large RNA transcript, 8.5 to 9.5 kilobases, possibly related to the fms oncogene in mouse, rat, and human tumor cells, has been described in the literature. However, the pSM3 fms probe used to detect this gene transcript contains a significant amount of the pol gene of the Susan McDonough strain of feline sarcoma virus from which it was derived. Using a fms probe which does not contain any viral pol sequences, no such "fms-related" transcripts were detected in cell lines previously reported to express the large transcripts. These cell lines did express a large 9.5-kilobase transcript which hybridized to a probe for murine leukemia virus. Partial sequence analysis of the 9.5-kilobase transcript detected with the pSM3 probe in transformed rat cells indicated sequence homology with AKV murine leukemia virus. Thus, the presence of large RNA transcripts, interpreted by us and others as being related to the oncogene fms, appears to be due to the expression of mouse retroviral sequences which hybridize to the viral pol region contained in the pSM3 fms probe. In the case of rat and human cells, such sequences appear to be acquired after the cells have been passaged in nude mice. These results should serve as a reminder of the important biohazard and data interpretation implications for investigations in which cells transfected with retroviral vector constructs are injected into nude mice, because rescue of the recombinant sequences in these cells could occur following infection by endogenous murine retroviral particles.

Role of DNA mismatch repair in the cytotoxicity of ionizing radiation.

The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.

Elevated mutant frequencies and increased C : G-->T : A transitions in Mlh1-/- versus Pms2-/- murine small intestinal epithelial cells.

Mutations in DNA mismatch repair (MMR) genes are associated with increased genomic instability and susceptibility to cancer. Mice rendered deficient in either Mlh1 or Pms2 as a result of gene targeting are prone to tumorigenesis, particularly, lymphomas. In addition, although Mlh1-/- mice also develop small intestinal adenomas and adenocarcinomas, Pms2-/- animals remain free of such tumors. To establish whether this phenotypic dichotomy might be associated with a quantitative and/or qualitative difference in genomic instability in these mice, we determined small intestinal epithelial cell DNA mutant frequency and mutation spectrum using a transgenic lambda-phage lacI reporter system. Mutant frequencies obtained from both Mlh1-/- and Pms2-/- mice revealed elevations of 18- and 13-fold, respectively, as compared to their wild-type littermates. Interestingly, we found that C : G-->T : A transitions were significantly elevated in Mlh1-/- mice, accounting in large measure for the 1.5-fold lacI mutant frequency increase seen in these animals. We hypothesize that the increased level of C : G-->T : A mutations may explain, in part, why Mlh1-/- mice, but not Pms2-/- mice, develop small intestinal tumors. Furthermore, the difference in the lacI mutational spectrum of Mlh1-/- and Pms2-/- mice suggests that other MutL-like heterodimers may play important roles in the repair of G : T mispairs arising within murine small intestinal epithelial cells.

Conserved synteny between the Fugu and human PTEN locus and the evolutionary conservation of vertebrate PTEN function.

Mutations of PTEN, which encodes a protein-tyrosine and lipid phosphatase, are prevalent in a variety of human cancers. The human genome 'draft' sequence still lacks organization and much of the PTEN and adjacent loci remain undefined. The pufferfish, Fugu rubripes, by virtue of having a compact genome represents an excellent template for rapid vertebrate gene discovery. Sequencing of 56 kb from the Fugu pten (fpten) locus identified four complete genes and one partial gene homologous to human genes. Genes neighboring fpten include a PAPS synthase (fpapss2) differentially expressed between non-metastatic/metastatic human carcinoma cell lines, an inositol phosphatase (fminpp1) and an omega class glutathione-S-transferase (fgsto). We have determined the order of human BAC clones at the hPTEN locus and that the locus contains hPAPSS2 and hMINPP1 genes oriented as are their Fugu orthologs. Although the human genes span 500 kb, the Fugu genes lie within only 22 kb due to the compressed intronic and intergenic regions that typify this genome. Interestingly, and providing striking evidence of regulatory element conservation between widely divergent vertebrate species, the compact 2.1 kb fpten promoter is active in human cells. Also, like hPTEN, fpten has a growth and tumor suppressor activity in human glioblastoma cells, demonstrating conservation of protein function.

Base transitions dominate the mutational spectrum of a transgenic reporter gene in MSH2 deficient mice.

Tumors derived from individuals with hereditary nonpolyposis colorectal cancer syndrome frequently demonstrate mutations in both alleles of hMSH2, a key gene in DNA mismatch repair (MMR). Sporadic tumors also frequently exhibit MMR deficiency. In keeping with the role of MMR in the maintenance of genome integrity, mice deficient in MSH2 via gene targeting demonstrate a high incidence of thymic lymphomas and small intestinal adenocarcinomas. To investigate the effects of MSH2 deficiency in normal tissues, mice containing a retrievable transgenic lacI reporter gene for mutation detection were crossed with MSH2-/- mice. Mice homozygous for MSH2 deficiency revealed 4.8, 11.0 and 15.2-fold elevations in spontaneous mutation frequency in DNA obtained from brain, small intestine, and thymus, respectively, as compared to heterozygous or wild-type mice. Mutations most frequently recovered from MSH2-/- mice were single base substitutions (77%), particularly base transitions (64%). Frameshifts occurred less frequently (19%) and fell within very short (3-5 bp) mononucleotide runs. Thus the number of key growth control genes potentially impacted by MMR deficiency extends beyond those containing repetitive sequences. These results highlight the capacity for MSH2 deficiency to serve as a potent driving force during the multi-step evolution of tumors.

Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hormone belonging to the glucagon superfamily of hormones. These hormones are known to play important roles in metabolism and growth. PACAP is a neuropeptide that causes accumulation of cAMP in a number of tissues and affects the secretion of other hormones, vasodilation, neural and immune functions, as well as the cell cycle. To determine whether PACAP is essential for survival and to evaluate its function(s), we have generated mice lacking the PACAP gene via homologous recombination. We found that most PACAP null mice died in the second postnatal week in a wasted state with microvesicular fat accumulation in liver, skeletal muscle, and heart. Gas chromatography-mass spectrometry showed that fatty acid beta-oxidation in liver mitochondria of PACAP(-/-) mice was not blocked based on the distribution of 3-hydroxy-fatty acids (C6-16) in the plasma. Instead, increased metabolic flux through the beta-oxidation pathway was suggested by the presence of ketosis. Also, serum triglycerides and cholesterol were significantly higher (2- to 3-fold) in PACAP null mice than littermates. In the fed state, both serum insulin and blood glucose were normal in 5-d-old null mice compared with their littermates. In contrast, fasted PACAP null pups had a significant increase in insulin, but a decrease in blood glucose compared with littermates. Glycogen in the liver was reduced. These results suggest PACAP is a critical hormonal regulator of lipid and carbohydrate metabolism.

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.

Construction of single-chain antibodies that bind an overlapping epitope of HIV-1 Nef.

The light and heavy chain variable regions of three mouse hybridoma cell lines (AG11, AE6 and EH1) that produce monoclonal antibodies against an overlapping epitope at the C-terminus of Nef were cloned. Sequence analysis of the light and heavy chain variable regions indicated that clones AG11 and AE6, but not EH1, were highly related. Single-chain antibodies were constructed from the cDNA clones of AG11 and EH1, and subcloned into an eukaryotic expressing vector with the green fluorescent protein as marker for expression. Such intracellular antibodies may provide a way in which to inhibit the function of Nef during HIV-1 infection of cells.

Cloning and chromosomal assignment of a widely expressed human receptor-like protein-tyrosine phosphatase.

Insight into the regulation of the actions of the protein-tyrosine kinases will be greatly facilitated by the full characterization of the family of protein-tyrosine phosphatases. A search for novel phosphatases resulted in the isolation of a cDNA, termed HLPR, encoding a member of the family of human receptor-like protein-tyrosine phosphatases: its cDNA sequence predicts a protein of 793 amino acids (unglycosylated Mr 87,500) and includes a 121 residue extracellular domain, a single transmembrane segment, and and two tandem intra-cytoplasmic catalytic domains. The HLPR genes is located on human chromosome 20, and the protein it encodes likely plays a fundamental role in the physiology of all cells as its expression appears to be ubiquitous.

Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells.

Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This colorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 x 10(4) lymphoid cells. The development of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity.

Mechanism of inhibition of protein-tyrosine phosphatases by disodium aurothiomalate.

Disodium aurothiomalate (AuTM) has been used successfully in the treatment of various autoimmune and inflammatory disorders; however, the molecular target(s) for this agent remains unknown. The aim of this study was to investigate whether the activity of CD45, a protein-tyrosine phosphatase (PTP, EC 3.1.3.48) essential for antigen-receptor-mediated lymphocyte signaling, was modified by AuTM exposure. The effects of AuTM on the activities of CD45 and other PTPs were monitored in vitro by a continuous assay using the substrate fluorescein diphosphate. In addition, the inhibition of PTP1B by AuTM was determined using a novel binding assay that employed an optical biosensor (BIAcore). The experimental results are summarized here: AuTM inhibited CD45 activity with an IC50 of 1.2 +/- 0.1 microM, and inhibition was competitive with substrate. The effect of AuTM, however, was not restricted to CD45, as the cytoplasmic PTP (PTP1B) was also inhibited, with an IC50 of 3.6 +/- 0.2 microM. AuTM also blocked the binding of GST-PTP1B to an immobilized active site inhibitor: a non-hydrolyzable difluorophosphonomethyl phenylalanine-containing biotinylated hexapeptide. AuTM-inhibited CD45 could be reactivated by the addition of excess dithiothreitol. These findings indicate that AuTM may interact with the essential active site cysteine residue involved in the catalytic mechanism of PTPs. Thus, it is possible that some of the cellular effects of gold result from the inhibition of these important cell signaling molecules.

Transfection of a factor-dependent cell line with the murine interleukin-3 (IL-3) cDNA results in autonomous growth and tumorigenesis.

The factor dependent murine myeloid line 32D c15 was transfected by electroporation with a murine interleukin-3 (IL-3) cDNA expression plasmid bearing the murine metallothionein-I promoter element. Factor-independent cell growth was readily obtained, and was shown to be accompanied by the production of biologically active IL-3. Three cell lines, growing autonomously and secreting IL-3 activity into their supernatants were established. S-1 nuclease analysis was employed to demonstrate that the introduced plasmid and not the endogenous IL-3 gene was the source of the IL-3 in one of these lines. The transfected IL-3 secreting cell lines, but not the parental factor-dependent 32D c15 cells, were uniformly able to induce tumors in syngeneic mice. These results indicate that the conversion to a malignant phenotype of a "partially transformed" cell line may be achieved by one additional dominant genetic event, such as the acquisition of autocrine growth factor secretion.

Human endothelial cells produce IL-6. Lack of responses to exogenous IL-6.

The interaction between human endothelial cells and leukocytes during immunological and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with lipopolysaccharide. The monokines, IL-1 and TNF-alpha, were potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. IFN gamma also was a strong stimulus of IL-6 production, but TGF-beta did not have an effect at doses modulating other endothelial cell functions. Endothelial cell IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell and hepatocyte stimulating factor. Endothelial IL-6 activity was neutralized by a specific antibody to IL-6 and it was shown by immunoprecipitation to be identical in size to human fibroblast-derived IL-6. IL-6 did not have a detectable effect on several endothelial cell functions, including proliferation, adherence of leukocytes, and synthesis of PGE2, TPA, and PAI-1. As IL-6 is probably an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.