Measurement of interleukin 6.

Interleukin 6 (IL-6) is a pluripotent cytokine with multiple effects on many different cell types. It is produced by a variety of cells in response to immunological and other stimuli. This unit describes a simple and sensitive assay for human, rat, rabbit, pig, and mouse IL-6, based on IL-6-dependent proliferation of a murine B cell hybridoma cell line, B9. Support protocols discuss maintenance of B9 cells, preparation of IL-6 standards, and production of IL-6-containing supernatant. In addition, IL-6 ELISA kits for the measurement of human or mouse IL-6 are available from a number of companies. These products are reliable, highly sensitive, and specific, and thus should be considered as an excellent (although more expensive) alternative, keeping in mind that bioactivity is not assessed with this approach.

Constitutive activation of STAT3 is associated with the acquisition of an interleukin 6-independent phenotype by murine plasmacytomas and hybridomas.

Interleukin 6 (IL-6), the major growth factor for myeloma cells, signals through the activation of signal transducers and activators of transcription (STAT) proteins. An important step in the malignant progression of murine plasmacytomas is the transition from dependence on IL-6 to a state of IL-6 independence. To elucidate the mechanism whereby IL-6 independence occurs, intracellular signaling events elicited by IL-6 in both IL-6-dependent and -independent plasmacytomas and hybridomas were compared. It was found that STAT3, a key molecule involved in IL-6 signaling, was constitutively activated and phosphorylated in IL-6-independent cell lines compared to the IL-6-dependent cells. Further comparison of upstream signaling pathways revealed that JAK-1 was constitutively present in anti-phosphotyrosine immunoprecipitates of IL-6-independent cells; gp130 was constitutively phosphorylated in a subset of IL-6-independent plasmacytomas, whereas other IL-6-independent lines showed no detectable gp130 phosphorylation in the absence of exogenous IL-6. Secretion of a factor capable of supporting the growth of IL-6-dependent cells was observed in one of the IL-6-independent plasmacytomas, but not in others, making an autocrine mechanism an unlikely explanation for IL-6 independence. These findings provide evidence that the constitutive activation of STAT3, either in the absence of detectable receptor-proximal events or associated with the concomitant activation of gp130, can contribute to the process of IL-6 independence.

Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7.

Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7. Both the response level (LPS inducibility) and the degree of inter-assay variation were evaluated for each transfection technique. The following methods were compared: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, SuperFect, Ca3(PO4)2 DNA co-precipitation, DEAE dextran-mediated transfection and electroporation. The transfected plasmid DNA included a luciferase reporter construct containing the junB minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galactosidase reporter construct under the adenovirus promoter and enhancer used as an internal control. Electroporation, followed by a resting period of 16-24 h before stimulation with LPS, had the highest inducibility of all methods. DEAE dextran and Ca3(PO4)2 precipitation showed the least and the greatest inter-assay variation, respectively. For all other methods, inter-assay variability fell within this range. The results presented may serve as both a general reference and a guide for reporter gene studies in this or other macrophage cell lines.

Regulation of actin cytoskeleton in lymphocytes: PKC-delta disrupts IL-3-induced membrane ruffles downstream of Rac1.

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL-3. Constitutively activated Rac1 restores the elongated cell phenotype to IL-3-starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL-3-induced assembly of membrane ruffles. Similar to the IL-3 withdrawal, phorbol 12-myristate 13-acetate (PMA) dissolves actin-formed membrane ruffles and rounds the cells in the presence of IL-3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL-3 the PMA-induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL-3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA-induced cell rounding can be reversed by the PKC-specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC-delta indicate that activation of PKC-delta but not other PKC isoforms is responsible for disruption of membrane ruffles.

Cross-talk between protein kinase C-alpha (PKC-alpha) and -delta (PKC-delta): PKC-alpha elevates the PKC-delta protein level, altering its mRNA transcription and degradation.

Studies utilizing the overexpression of individual isoforms indicated that both PKC-alpha and -delta promote a number of biological effects, including inhibition of DNA synthesis associated with rearrangements of the actin cytoskeleton in the murine B-cell lymphoma (Baf3), differentiation of the murine promyelocyte line 32D, and activation of MAP kinase in CHO fibroblasts. We postulated that these results reflect some form of cross-regulation between PKC-alpha and -delta rather than their functional redundancy. In this report, we show that overexpression of PKC-alpha in Baf3 and 32D leads to an elevation of the endogenous PKC-delta mRNA and protein levels. The elevated steady-state PKC-delta mRNA level results from a combination of increased PKC-delta transcription and mRNA stability. Upregulation of PKC-delta mRNA by PKC-alpha occurs even after a selective depletion of the PKC-delta protein. In addition, phorbol ester-induced elevation of PKC-delta mRNA and protein levels can be prevented by the PKC inhibitor GF109203X, an indication of the requirement for PKC kinase activity. Inhibition of new protein synthesis by cycloheximide showed that upregulation of PKC-delta mRNA, as opposed to delayed downregulation of the PKC-delta protein, is primarily responsible for the accumulation of this isoform by PKC-alpha. In parental Baf3 and 32D cells and PKC-alpha overexpressers, PKC-alpha and PKC-delta are uniquely involved in cross-regulation, while PKC-epsilon, PKC-eta, and PKC-mu are not.

Mechanism of apoptosis suppression by phorbol ester in IL-6-starved murine plasmacytomas: role of PKC modulation and cell cycle.

We show here that the mode of cell death in IL-6-starved T1165 and T1198 plasmacytoma cell lines is apoptosis, and that it can be suppressed by phorbol ester (PMA) treatment in a protein kinase C (PKC)-mediated process that involves alpha and/or delta isozymes. PMA-induced PKC activation, but not the depletion that follows it, participates in the suppression of apoptosis. Extended PKC activation is necessary but not sufficient for the apoptosis suppression. In addition, the cells must be in a "competent" state, which appears not to be determined by PKC. We observed two points of "competence" during the time between withdrawal of IL-6 and the start of massive cell death: one, immediately after withdrawal, and another, just before onset of apoptosis, at the time corresponding to maximal accumulation of cells in a G0/G1 block imposed by IL-6 withdrawal. Treatment with PMA and other PKC activators resulted in a shift of the cell population to S phase, lifting the G0/G1 block. We propose a model in which cells are rescued in a certain stage of the G1 phase of cell cycle. Death suppression occurs when a transient PMA-induced PKC activation occurs when a significant number of cells are in this part of G1, allowing them to pass the restriction point safely without initiating the cell death program.

An acute phase response factor/NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma.

The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect, we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and evaluated the IL-6 inducibility of these sequences by transient expression in an IL-6-dependent plasmacytoma cell line. Although a 6.5-kilobase fragment of upstream flanking sequence did not increase the IL-6 inducibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down-stream flanking sequence that both up-regulates the promoter and confers inducibility by IL-6. Point mutation of an acute phase response factor (APRF) site within this region significantly reduced up-regulation of the promoter in cells grown continuously in IL-6, as well as inducibility upon restimulation of cells with IL-6 after withdrawal from the growth factor. Point mutation of an NF-kappa B site sharing five nucleotides with the APRF site reduced up-regulation of the promoter but not inducibility by IL-6, whereas mutation of two other NF-kappa B sites in the 222-base pair fragment had no effect on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and constitutive binding of NF-kappa B p65 to the APRF/NF-kappa B site.

Suramin blocks the binding of interleukin-1 to its receptor and neutralizes IL-1 biological activities.

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 alpha to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 alpha and EL-4.6.1 cells confirms that suramin inhibits the binding of the ligand to the 80 kDa IL-1 type I receptor. In contrast, suramin fails to displace significantly prebound IL-1. In a cell-free system, suramin prevents the binding of IL-1 alpha and IL-1 beta to murine and human recombinant soluble type I IL-1 receptors. For example, the IC50 for suramin inhibiting IL-1 alpha and IL-1 beta binding to soluble human IL-1 receptor were 204 microM and 186 microM, respectively. The suramin analogues, NF-058 and NF-103 (which bear the same number of sulfate groups as suramin), are between three- and ten-fold less active than suramin in inhibiting IL-1 binding to EL-4.6.1 cells, and to recombinant soluble IL-1 receptor. Furthermore, in a dose-dependent manner suramin prevents several IL-1 mediated biological responses, including thymocyte proliferation, PGE-2 synthesis and IL-6 production. The inhibitory effect of the drug can be significantly reversed by the addition of excess cytokine. Taken together, the results indicate that suramin is a competitive IL-1 receptor antagonist. Because IL-1 participates in a broad range of immunological and inflammatory functions, the data suggest that suramin administration may influence important activities beyond those associated strictly with tumor inhibition.

Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo.

Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia. Recently, the involvement of interleukin-6 (IL-6) in the wasting of colon-26 adenocarcinoma-bearing mice was demonstrated. The research presented here establishes an anticachectic role for the experimental drug suramin, since it partially blocks (up to 60%) the catabolic effects associated with the growth of this tumor in vivo. Suramin prevents the binding of IL-6 to its cell surface receptor subunits, as demonstrated by radioreceptor binding assay and affinity crosslinking experiments. Furthermore, the uptake of radioactive IL-6 by the liver is significantly reduced in suramin-treated mice. On the other hand, the drug is approximately 10-fold less potent in inhibiting the binding of tumor necrosis factor-alpha to indicator cell line in vitro and fails to block liver uptake of this cytokine in vivo. Collectively, these results suggest that suramin inhibits cancer-associated wasting, in part by interfering with the binding of IL-6 to its receptor. Whether suramin inhibits the action of other factors/cytokines that may also participate in colon-26-mediated cachexia is not yet known.

Direct association of interleukin-6 with a 130-kDa component of the interleukin-6 receptor system.

Affinity cross-linking of membrane bound 125I-interleukin-6 (IL-6) on several cell lines revealed a three-band pattern of IL-6-containing cross-linked complexes with molecular masses of 100, 120, and 150 kDa. To identify the membrane components that were associated with IL-6 in the three complexes, we employed the Denny-Jaffe reagent, a heterobifunctional, cleavable cross-linker that allows the transfer of 125I from the ligand to its receptor. Samples cross-linked with Denny-Jaffe reagent were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis in which the cross-linker was cleaved prior to the second dimension. This analysis revealed that IL-6 directly associates with a 130-kDa membrane protein thus allowing the formation of the 150-kDa complex. In addition, both the 100- and 120-kDa cross-linked complexes were shown to include an 80-kDa membrane glycoprotein associated with one and two IL-6 molecules, respectively.

Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia.

To investigate whether interleukin 6 (IL-6) might be a potential mediator of the depleted fat reserves observed in malignancy-associated cachexia, we measured lipoprotein lipase (LPL) activity in adipose tissue of mice after administration of IL-6 or tumor necrosis factor and in cultured adipocytes after addition of these cytokines. Injection of IL-6 i.p. reduced adipose tissue LPL activity by 53% within 4.5 to 5.5 h. Injection of tumor necrosis factor elevated serum IL-6 levels and reduced adipose tissue LPL activity by 70%. Both human and murine IL-6 reduced heparin-releasable LPL activity in 3T3-L1 adipocytes in a dose-dependent manner; half-maximal inhibition of LPL activity was achieved with 5000 hybridoma growth factor units/ml. Thus, IL-6 reduces adipose LPL activity and may contribute to the loss of body fat stores associated with some cases of cancer cachexia. Since tumor necrosis factor increases circulating IL-6, some of its effects may be mediated or potentiated by IL-6.

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function.

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.

Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266.

It has been reported recently that freshly isolated human myeloma cell cultures proliferate in response to added interleukin-6 (IL-6). Endogenous levels of IL-6 found in the same cultures suggested that an autocrine growth loop may contribute to cell growth. However, the lack of homogenous cell populations in primary myeloma cultures has made it difficult to distinguish between paracrine and autocrine growth mechanisms. To precisely address the autocrine growth issue we have evaluated the growth of the human myeloma cell line, U266. We have found that a neutralizing anti-IL-6 monoclonal antibody can inhibit U266 proliferation. Furthermore, the addition of IL-6 antisense oligonucleotides also inhibits U266 proliferation. These effects are reversed by adding IL-6, suggesting the presence of an autocrine loop. Using bioassays with two different IL-6-dependent cell lines, we were able to detect IL-6 in concentrated U266 supernatants. IL-6 mRNA was detected by polymerase chain reaction amplification of cDNA. Cell cycle parameter analysis shows that IL-6 acts to release a block in G1. Taken together these results present conclusive evidence for IL-6-mediated autocrine growth in the U266 human myeloma cell line.

Expression of the interleukin 6 receptor and interleukin 6 in prostate carcinoma cells.

We have probed for the presence of interleukin 6 (IL6) receptors in prostatic carcinoma cell lines (LNCaP, DU 145, and PC3) by examining their sensitivity to the cytotoxic effects of a chimeric toxin composed of IL6 and Pseudomonas exotoxin (PE). All three cell lines were killed by IL6-PE66(4)Glu, a version of IL6-PE in which the binding domain of native PE has been mutated to debilitate PE binding to its own receptor. This cytotoxic activity confirmed the presence of IL6 receptors on prostatic carcinoma cells. We have measured the number of IL6 receptors found on these cells and have further determined that they secrete IL6. These data provide evidence that IL6 and its receptor may play an important role in human prostate cancer.

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.

T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells.

IL-6 (formerly PCTGF, HP-1, BSF-2, HGF, IFN-beta 2, 26 kDa) is a recently defined lymphokine demonstrating activity on multiple cell types, including hepatocytes, thymocytes, T cells, plasmacytomas, and B cells. The biologic effects of IL-6 on lymphocytes, particularly B cells, suggest this factor may be involved in the regulation of normal immune responses. Accordingly, we have investigated the role of IL-6 in Ag-specific responses of B cells from both naive and Ag-primed mice. When Ag-primed splenic T cells were used as a source of help, naive (primary) B cell responses specific for the hemagglutinin molecule of the influenza A virus (PR8) were fully inhibited by the addition of an anti-IL-6 antiserum, and are thus IL-6 dependent. In contrast, secondary B cell responses were essentially IL-6 independent, being unaffected by this antiserum even at concentrations 10-fold higher than required to completely inhibit primary responses. This differential IL-6 requirement was further investigated by using a panel of hemagglutinin molecule-specific Th clones. Consistent with the above findings, a Th1 clone secreting biologically active IL-6 enables antibody secretion by both primary and secondary B cells, whereas Th1 clones that do not produce IL-6 support secondary responses, but fail to help primary B cell responses unless exogenous IL-6 is added. These results provide the first instance of differential lymphokine requirements among primary vs secondary B cell responses, and suggest T cell-derived IL-6 plays a critical role during the regulation of humoral immune responses. Moreover, functionally distinct Th1 clones were identified that differed in IL-6 secretion and their corresponding ability to induce Ig secretion by primary and secondary B cells.

Interleukin 6 (interferon beta 2) and interferon alpha/beta present in postendotoxin serum induce differentiation of murine M1 myeloid leukemia cells.

Serum from lipopolysaccharide-treated mice (postendotoxin serum, PES) induces the differentiation of M1 myeloid leukemia cells into mature macrophages, as well as supporting the proliferation of the interleukin 6 (IL6)-dependent B9 hybridoma cells. The kinetics of appearance of these two activities in PES were identical. To determine whether these two activities are due to the presence of the same substance, we tested whether anti-IL6 antibodies could neutralize the differentiation-inducing activity of PES. We found that anti-IL6 antibodies completely neutralized the proliferation of B9 cells and resulted in a 60% neutralization of the differentiation-inducing activity of PES. Anti-interferon alpha/beta (INF alpha/beta) antibodies neutralized 70% of the differentiation-inducing activity of PES. These data suggest that the differentiation-inducing activity of PES is not limited to IL6, and that PES contains additional factors such as INF alpha/beta that are capable of inducing differentiation of M1 cells.

IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis.

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells. rIL-1 alpha or purified IL-6 only slightly enhanced synthesis of IgM over minimal baseline levels in Peyer's patch T cell-depleted cell cultures; however, when IL-6 was added to cultures also containing rIL-1, IgM synthesis was very substantially increased. In addition, rIL-5 alone gave rise to a modest increase in IgM synthesis and its effect was not enhanced by either rIL-1 or IL-6. IgG production (mainly IgG3) followed a similar pattern. In contrast, IgA production was only modestly increased above baseline by rIL-1, rIL-5, or IL-6 alone or by rIL-1 and IL-6 in combination, but was greatly increased by rIL-5 and IL-6 in combination. The effect of IL-6 on Ig synthesis in the above studies was not due to an effect on cell proliferation. In summary, these data indicate that B cells differ in respect to the cytokines supporting maximal terminal differentiation and thus the class of Ig produced may depend on the presence of a particular combination of cytokines and lymphokines.

Murine thymocytes proliferate in direct response to interleukin-7.

The ability of interleukin-7 (IL-7) to stimulate murine thymocyte proliferation was investigated. IL-7, either alone or in concert with lectin, induced proliferation of adult thymocytes as well as day 13 fetal and adult CD4-/CD8-thymocytes. The IL-7-induced proliferative response of unfractionated thymocytes could not be inhibited by antibodies to IL-2, or IL-4, IL-6, or the IL-2 receptor. In addition, IL-2, IL-4, and IL-6 were not produced by thymocytes activated with IL-7, as judged by the absence of biologically active cytokine in IL-7-stimulated culture supernatants. IL-7 could act in concert with IL-2 and IL-4 or with IL-4 to enhance the proliferative response of thymocyte cultures. Thus, IL-7 may cause proliferation of thymocytes directly, not indirectly, through production of IL-2, IL-4, or IL-6. IL-7 may then play a significant role in differentiation of T lymphocytes.

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice.

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.