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Many plasmacytomas arising in BALB/c mice require a specific, macrophage-derived growth factor in order to proliferate in vitro. Since transferrin receptor expression is normally regulated by tissue-specific growth factors and because expression of these receptors is required for cell proliferation, we examined the interaction of plasmacytoma growth factor (PCT-GF) on transferrin receptor expression and cell cycle progression in several PCT-GF-dependent and independent plasmacytoma cell lines maintained in vitro. We found that removal of PCT-GF results in a rapid and specific loss of transferrin receptor expression with concomitant G1 arrest in early G1. The time required for G1 arrest to become maximal correlates closely to the initial level of surface transferrin receptor expression and the rate of decay following removal of PCT-GF. The calcium channel blocker diltiazem interferes with the ability of PCT-GF to maintain transferrin receptor expression in PCT-GF-dependent cell lines and causes a G1 arrest of the cell population. When added to a PCT-GF-independent cell line, diltiazem also inhibited transferrin receptor expression and caused G1 arrest. Thus, both PCT-GF-dependent and -independent plasmacytoma cell lines require transferrin receptor expression for growth. In factor dependent cell lines, transferrin receptor expression requires exogenous PCT-GF, while in factor-independent cells, transferrin receptor expression is constitutive. In both cell types, intracellular calcium levels may play a role in receptor expression.

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.

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.

Many plasmacytomas arising in BALB/c mice are dependent upon a specific, macrophage-derived plasmacytoma growth factor for survival and proliferation in vitro. Adherent cells taken from the peritoneal oil granuloma in which the early plasmacytomas arise and proliferate produce 50 times more PCT-GF activity in vitro than do normal peritoneal cells, thus suggesting a possible in vivo role for PCT-GF. Purification and amino acid sequencing of PCT-GF derived from the murine macrophage cell line, P388D1, have identified a 23 kDa protein with a unique NH2-terminal sequence. This molecule is now known as murine IL6. As part of the characterization of murine Il-6, genomic sequences have been localized to the proximal end of mouse chromosome 5 via Southern analysis of restriction fragment length polymorphisms. The removal of IL6 from IL6-dependent PCT cell lines results in a growth arrest in early G1. This is accompanied by a rapid and specific loss of transferrin receptor expression and results in eventual cell death. It appears that the response to IL6 is at least partially dependent on Ca++ because functional Ca++ channels are necessary for the PCT cells to pass through G1 and to maintain transferrin receptor expression.

It was recently shown that T cells, macrophages and fibroblasts produce growth factors for B cell hybridomas and plasmacytomas. These factors were subsequently identified as members of a new family of cytokines on the basis of NH2-terminal amino acid sequence analyses. Using T cell-derived interleukin-HP1 (HP1), purified to homogeneity, as the prototype of this family, we examined the effects of these molecules in conventional polyclonal B cell activation assays with anti-immunoglobulin antibodies or dextran sulfate as co-stimulators. In the absence of other cytokines, the only significant effect of HP1 was to stimulate moderately the proliferation of anti-immunoglobulin-activated B cells. By contrast, in conjunction with interleukin 1, HP1 became a major growth and differentiation factor not only for B cells activated with anti-immunoglobulin antibodies but also for dextran sulfate-stimulated and even for unstimulated B cells. In fact, with respect to cell proliferation or IgM synthesis, the IL 1-HP1 combination proved to be equivalent to B cell stimulatory factors like IL 4 or IL 5. This B cell stimulatory activity was not due to the presence of a contaminant in the HP1 preparation because it was also observed with purified plasmacytoma growth factors derived from macrophages and fibroblasts, and could be inhibited by a monoclonal anti-HP1 antibody.

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.

IL 1 and IL 6 share a number of biological activities, including induction of fever, neutrophilia and acute phase response, and IL 1 induces IL 6 production by fibroblasts and macrophages. Therefore, it was proposed that IL 6 mediates many of the activities of IL 1. To test this hypothesis in vivo, we assessed induction of IL 6 following IL 1 alpha administration to mice and tested IL 6 for radioprotection and induction of early (CSF) and late (fibrinogen and SAA) acute phase reactants. IL 1 alpha given to mice ip induced, in a dose dependent manner, detectable IL 6 in circulation, with maximal titers at 2-4 hrs. However, unlike IL 1 which is 10-1000 ng/mouse of human recombinant IL 6 did not result in increased survival of mice following lethal irradiation. In fact, such treatment given 20 hrs before LD50/30 doses of radiation resulted in reduced survival of mice. However, IL 6 augmented the radioprotective effect of IL 1. IL 1 in doses above 10 ng/mouse induced within 2 to 6 hrs a dose dependent increase in CSF in circulation, but IL 6 did not induce detectable levels of CSF at 2, 6 and 20 hrs after administration. Administration of IL 6 to mice produced a dose dependent increase in circulating fibrinogen and SAA. Similarly, administration of IL 1 resulted in much greater increases in levels of fibrinogen and SAA. Therefore, IL 1 is a more effective inducer of fibrinogen and SAA in mice than is IL 6. Although definitive conclusions concerning the relative roles for IL 1 and IL 6 in vivo will await availability of anti IL 1 and anti-IL 6 antibodies, our data do not support the suggestion that the above IL 1 effects can be attributed solely to IL 6.

The activation of highly purified murine peripheral T cells in vitro by Con A is dependent on a co-stimulatory signal that is not IL-1 or IL-2. Previous evidence has demonstrated that the recently defined lymphokine IL-6 could provide costimulatory activity for purified T cells cultured with Con A. In this report we demonstrate that IL-7 also has potent co-stimulatory activity for purified murine T cells, as well as its previously described ability to support the growth of pre-B cells in Witte-Whitlock cultures. When rIL-7 was added to cultures of purified T cells together with Con A, it induced the expression of IL-2 receptors, IL-2 production, and consequently proliferation. In addition, IL-7 exhibited the same magnitude of activity in this assay as IL-6. Also, anti-IL-6 antibody which inhibited the IL-6-induced response had no effect on the IL-7 response. Thus, IL-7 does not act by inducing IL-6. These results demonstrate that IL-7, a potent growth stimulus for pre-B cells, also has a role in T cell activation.

Murine Il-6 cDNAs were isolated by cross-hybridization with a human IL-6 cDNA from an IL-1 activated bone marrow stromal cell line (W20). Mouse-hamster somatic cell hybrids were utilized to localize murine Il-6 to chromosome 5. Genetic mapping with respect to En-2, AlbH, and Gus in backcross progeny from an interspecific mating between C57BL/6J and Mus spretus positioned Il-6 3 cM distal to En-2. The syntenic relationships of Il-6 and En-2 in mouse and man, as well as the potential role of IL-6 in tumorigenesis, are discussed.