Native human IL-1 beta and IL-1 alpha stimulated prostaglandin E2 secretion by human embryonic lung fibroblasts at half-maximal concentrations of 3 +/- 1.2 pM (+/- SEM) and 10 +/- 2.3 pM, respectively. In contrast to the 20-50-fold lower affinities previously found for IL-1-R on 3T3 cells as well as murine and human lymphoblastoid lines, monoiodo 125I-IL-1 beta bound to normal human fibroblasts with a Kd of 8.4 +/- 4.1 pM in direct binding experiments, and with a Ki of 11.2 +/- 2.8 pM in competitive binding experiments. IL-1 alpha bound to the receptor identified by 125I-IL-1 beta with a Ki of 50 +/- 18 pM. The receptor exhibited homogeneous affinity for IL-1 beta or IL-1 alpha. The receptor did not recognize IL-2, IFN-gamma, tumor necrosis factor alpha, a functionally related monokine, or bovine acidic fibroblast growth factor, a structurally related mediator. Comparison of the biological response curves and binding curves obtained for IL-1 alpha and IL-1 beta showed that they were parallel and that 10-15% occupancy of the estimated 3,000 sites by either species of IL-1 was sufficient to give half-maximal stimulation of prostaglandin E2 secretion. Thus, the amount of apparent signal amplification observed on fibroblasts was considerably lower than the 100-100,000 fold amplification previously reported for lymphoid lines. Crosslinking experiments revealed a major band with a corrected molecular mass of approximately 80 kD and a minor band of approximately 200 kD. Labeling of these bands was blocked by IL-1 beta and IL-1 alpha but not by IL-2, IFN-gamma, or tumor necrosis factor alpha. These results demonstrate that normal human embryonic lung fibroblasts bear IL-1-R of sufficiently high affinity to mediate their biological responsiveness to low picomolar concentrations of IL-1 beta and IL-1 alpha and are consistent with the existence of a single receptor mediating the biological properties of both human IL-1 species.

In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (ILI-) a and ft in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliberation assay, and in an assay measuring stimulation of prostalandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-I concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-la and II-10 are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.

No abstract

The term IL-1 has been used to describe a family of monocyte derived polypeptides that have immunomodulatory and proinflammatory biological properties (1, 2). While most studies have focused on IL-1 activity that is found in monocyte/macrophage culture supernatants, early studies showed that high amounts of activity could also be recovered from cell lysates. These studies suggested that IL-1 may accumulate within mononuclear phagocytes in a precursor form before release into the surrounding medium (3-5).Recently it has been shown that there are at least two distinct human IL-1 molecules termed IL-la and IL-13. Separate mRNAs for these IL-1 species each code for a precursor of ~31 kD, which is subsequently processed by still undefined mechanisms to a "mature" molecule of ~17.5 kD (6). The absence of a typical hydrophobic leader sequence in either of the IL-1 molecules identified to date suggests that they may not be typical secretory proteins. A 33-kD precursor for murine IL-1, which is ~62% homologous to human IL-la (6), has been documented at the protein level in cell lysates of peritoneal macrophages, as well as in lysates of the murine macrophage line P388D~ (7). Comparable studies have not yet been reported for human monocytes and macrophages.The successful development of highly specific heterologous antisera (8) for the species of human IL-1 having a pI of 6.8 (IL-18) (6, 9) has allowed us to identify its precursor in the cytoplasm of activated human monocytes. In the present report, the intracellular accumulation of IL-1 is documented using indirect immunofluorescence performed on fixed and permeabilized cells, and immunoblot analysis of cell lysates. These studies show that it is possible to identify IL-1-producing cells at the light microscopic level and suggest that anti-IL-1 antibodies may be useful for the in situ localization of IL-1 production in inflammed tissue. Materials and MethodsAntisera. A detailed description of the peptides used, as well as the methodology for raising and characterizing antisera, is presented elsewhere (8). Briefly, peptides Ala ~7-Asp l 2s (p 117 -128) and of the amino acid sequence derived from I L-1 cDNA (6, 10) were synthesized using standard solid phase methodology (11), coupled to KLH with the heterobifunctional reagent m-maleimidobenzyl-N-hydroxysuccinimide

In this report the binding of recombinant human interleukins la and 1,8 ) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-la required for half-maximal stimulation of PGE2