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A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.

Stress reactions exist in many conditions in which plasma interleukin 6 (IL-6) is elevated. Examples are burns and sepsis. In these situations fever is often present. These stress situations are always accompanied with high levels of adrenalin and corticosteroids. These hormones, especially when given together, elicit a definite response of acute phase proteins in normal rats. In two stress models, (i) laparotomy and (ii) fever induced by administration of PGE2 in the lateral intracerebral ventricle, we observed a rise of adrenalin and corticosteron followed by an elevated level of plasma IL-6. Therefore, we studied the effect of adrenalin and corticosteron on the plasma level of IL-6. Adrenalin evokes high levels of IL-6, and this effect can be blocked by propranolol. When IL-6 release is blocked in this way, the response of alpha 2 macroglobulin and the cysteine protease inhibitor, both fast-reacting acute phase proteins in rat, is strongly depressed. Isoprenalin, an adreno beta 2 agonist, also causes very high levels of IL-6, indicating that the release of IL-6 can be mediated by an adreno beta 2 receptor whose presence has been demonstrated in monocytic cells. The results suggest a relation between stress situations and IL-6 and may be another factor besides the presence of endotoxins, virus, etc. explaining the high levels of IL-6 observed in many serious clinical situations.

Interleukin-6 (IL-6) is produced by various cell types, including monocytes, fibroblasts, and endothelial cells. IL-6 has also been detected in the urine of normal and renal transplant patients. Thus, the possible production of this cytokine by glomeruli and mesangial cells was investigated. Rat glomeruli were obtained by serial sieving of cortical homogenates of blood-free kidneys. Mesangial cells were obtained from the glomeruli and cultured under standard methods in RPMI 1640 medium containing 15% fetal calf serum. Glomeruli or confluent monolayers cells were then incubated in RPMI 1640 for 18 hr, in the presence or not of tumor necrosis factor-alpha (TNF alpha), lipopolysaccharide (LPS), or platelet-activating factor (PAF). IL-6 activity was measured using the IL-6-dependent cell line subclone (B 9-9) and expressed with respect to a standard curve established with recombinant IL-6. Glomeruli generate IL-6 upon TNF alpha (100 ng/ml) and LPS (1 microgram/ml), 11,500 +/- 3000 and 22,000 +/- 7500 U/ml, respectively. Nonstimulated mesangial cells produced 50 +/- 5 U/ml (mean +/- SEM, n = 4) of IL-6. TNF alpha (1 ng/ml) and LPS (1 microgram/ml) induced the production of 800 +/- 90 and 40,000 +/- 5000 U/ml, respectively (n = 4). In contrast, PAF (0.1 nM-1 microM) did not increase IL-6 production from glomeruli or mesangial cells. These results demonstrate that renal cells spontaneously generate minimal amounts of IL-6 and that this production is significantly increased by TNF alpha or LPS. A synergy between LPS and TNF alpha was induced in glomerular cells with 10 ng/ml of TNF alpha and graded concentrations of LPS. Thus, the production of IL-6 by glomerular cells and its modulation by other cytokines or endotoxins may play a role in the local immunological processes leading to immune glomerular diseases.

Stimulation of tonsils or peripheral blood lymphocytes with either pokeweed mitogen (PWM) or interleukin-2 (IL-2) results in significant IgM production. However, the combination of PWM and IL-2 shows a synergistic IgM synthesis in tonsil cells, whereas the secretion in peripheral blood cells is diminished compared to activation with either stimulus alone. The heavy-chain isotype distribution does not significantly change in the two cell types when stimulated with either PWM, IL-2, or the combination. The Ig synthesis by tonsil cells to PWM or IL-2 drastically increases in the presence of monocytes, but the response to PWM + IL-2 together is not affected. The reason for the synergistic IgM production to PWM + IL-2 in tonsil cells is the relative lack of monocytes and the low number of T8+-suppressor cells. The decreased IgM production in peripheral blood cells after stimulation with both PWM and IL-2 is due to the presence of T8+ cells. These data show that the monocyte-dependent IL-2 production is an essential step in PWM-induced Ig secretion; the subsequent induction of the T-cell-helper signal for B-cell differentiation is determined by the balance between the strength of the stimulus for T cells and the T4 T8 ratio. Hence, these parameters should be included when the B-cell function is studied in vitro with the use of PWM and IL-2.

Mononuclear cells of five patients with B-prolymphocytic leukemia were tested for their functional capacities in vitro after stimulation. Leukemic B cells from these patients have a mature phenotypic marker profile and can be obtained in high numbers from peripheral blood. B cells of all five patients proliferated in response to phorbol myristate acetate. In one patient, this proliferation was strongly enhanced by the addition of T-cell-conditioned medium. Conditioned medium by itself or interleukin 2 in the presence or absence of the phorbol ester was not active. This enabled us to develop an assay for a B-cell growth factor different from interleukin 2. Next to their proliferative capacities, we found that malignant B cells of three of the five patients secreted large amounts of IgM when stimulated with pokeweed mitogen or interleukin 2 in the presence of allogeneic T cells. In the absence of these allogeneic cells, neither pokeweed mitogen nor interleukin 2 had any effect. However, T-cell-conditioned medium as well as the supernatants of two T-cell hybridomas induced strong IgM production in the absence of T cells. Thus, neoplastic B cells of some patients with B-prolymphocytic leukemia can be stimulated to proliferation and differentiation in vitro and can be used as an assay and model system to study the effect of T cells and/or T-cell factors in human B-cell activation.

The ability of Escherichia coli-derived lipopolysaccharide (LPS), recombinant (r) interleukin 1-beta (rIL-1 beta), and r murine tumor necrosis factor-alpha (rMuTNF-alpha) to induce interleukin 6 (IL-6) production in vivo was investigated. Peak serum IL-6 concentration was attained after 2 hr of LPS injection into mice. The coinjection of antiserum against rMuTNF-alpha with LPS resulted in a reduction of the induced serum IL-6 level, indicating the involvement of endogenous TNF-alpha in LPS induction of IL-6. Recombinant IL-1 beta and rMuTNF-alpha injected directly caused the production of substantial amounts of IL-6 within 30 min. The injection of a combination of rIL-1 beta and rTNF-alpha induced a significantly greater level of IL-6 than either agent alone. The greater level of serum IL-6 was associated with hypothermia and an increased lethality among mice injected with both cytokines. These data demonstrate the abilities of IL-1 beta and TNF-alpha to induce IL-6 production in vivo and indicate that LPS induction of IL-6 may be mediated, at least partially, through TNF-alpha action. The data describe a new in vivo biologic activity shared between IL-1 beta and TNF-alpha and suggest that IL-6 may be an important effector in the manifestation of TNF-alpha and IL-1 beta actions in vivo.