rHuIL-1 Alpha Research Articles

The effects of the phospholipase a2 inhibitor, manoalide, on cartilage degradation, stromelysin expression, and synovial fluid cell count induced by intraarticular injection of human recombinant interleukin‐1α in the rabbit

To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.

Stem cell responses in myelosuppressed mice following sequential treatment with recombinant human interleukin 1 (rHuIL‐1), recombinant murine interleukin 3 (rMuIL‐3) and recombinant human macrophage colony‐stimulating factor (rHuM‐CSF)

In vivo, recombinant human interleukin 1 alpha (rHuIL-1 alpha) + recombinant human macrophage colony-stimulating factor (rHuM-CSF) (IL-1 + M-CSF) effectively serves as a rescue agent for myelosuppression by enhancing the recovery of hematopoietic stem cell (HSC) subpopulations following treatment with 5-fluorouracil (5-FU). Because in vitro studies have suggested that hematopoietic recovery in 5-FU-treated bone marrow (FUBM) may proceed from a 5-FU resistant, (IL-1 + IL-3 + M-CSF-responsive) high proliferative potential HSC subpopulation of colony forming cells (HPP-CFC), studies were carried out to determine whether the addition of recombinant murine interleukin 3 (rMuIL-3) (IL-3) to either IL-1 or IL-1 + M-CSF would further enhance the recovery of HSC subpopulations in myelosuppressed C57Bl/6 mice. With the exception of the HPP-CFC, IL-3 dampened, rather than enhanced, the accelerated recovery of 8 d and 12 d colony forming units-spleen (8 d and 12 d CFU-S) and the committed macrophage progenitor (CFU-M) associated with in vivo treatment with IL-1 alone. Similarly, IL-3 interfered with the enhanced recovery of those HSC subpopulations in FUBM influenced by the synergistic interaction of IL-1 + M-CSF. This interference, however, was observed only when the rMuIL-3 was administered on day 2 or 3 of a four-day treatment with IL-1 + M-CSF. There was, however, no evidence that IL-3 exerted a negative influence on the restoration of granulocytes in the myelosuppressed animals. Moreover, sequencing studies provided data suggesting that the dampening effects of IL-3 on the synergistic interaction of IL-1 + M-CSF resulted from both an enhanced differentiation of the more primitive HSC subpopulations and a significant, but preferential, mobilization of the more mature 8 d CFU-S and CFU-M to extramedullary organs and that the mobilization of these more mature HSC subpopulations was temporally linked to their generation from the recovering HPP-CFC and 12 d CFU-S subpopulations.

Cryoprotective effect of gelatin and albumin on recombinant human tumor necrosis factor liposome.

A search for a cryoprotective agent of a liposome containing recombinant human tumor necrosis factor (rHuTNF) was undertaken. Various additives were added and liposomes were freeze-thawed. As a result of these studies, albumin and gelatin were found to suppress the leakage of rHuTNF from liposome. This cryoprotective effect was only observed when the additives were added to the inside of the liposomal membrane; their addition to the outside of the membrane stimulated the leakage. This characteristic was clearly different from well-known cryoprotective agents, such as saccharides which have the cryoprotective effect on both sides of the liposomal membrane. Although this cryoprotective effect was also observed for recombinant human interleukin-1 alpha (rHuIL-1 alpha), it was not observed for fluorescein sodium. These effects of gelatin and albumin were restricted to macromolecular proteins such as rHuTNF and rHuIL-1 alpha, but both substances were very effective cryoprotectants for a liposome containing protein.

Effect of IL-1 on experimental bone/bone-marrow metastases.

Bone metastasis is a common event and a major cause of morbidity in cancer patients. The hematopoietic marrow of the bones, rather than the bone tissue per se, is the target organ in bone metastasis. In the bone marrow, IL-1 induces the release of hematopoietic growth factors that may affect tumor-cell growth. We treated groups of mice with rhuIL-1 alpha to examine its role in the establishment of experimental bone/bone-marrow metastasis. We found that injection of 2 micrograms of rhuIL-1 alpha 24 hr prior to, simultaneously with or 24 hr after the injection of 10(4) B16 melanoma cells into the left cardiac ventricle of mice resulted in a 2-fold increase in the average number of colonized bones per mouse. GM-CSF is produced by bone-marrow stromal cells in response to IL-1, and its receptor has been found on tumor cells, including melanoma cells. However, the administration of rmuGM-CSF to mice by either multiple injections or continuous infusion did not affect the number of colonized bones. Many of the biologic effects of IL-1 are mediated by prostaglandins. Treatment of mice with 100 micrograms of indomethacin, a potent inhibitor of prostaglandin synthesis, prior to the injection of rhuIL-1 alpha, prevented the increase in number of bone metastases. To determine whether constitutive productions of IL-1 and/or prostaglandins are involved in the pathogenesis of bone/bone marrow metastasis, we treated mice with antimouse IL-1 alpha neutralizing antibodies, rhuIRAP (an inhibitor of IL-1 activity) or indomethacin. We found no difference in the average number of colonized bones per mouse between treated and control mice. We conclude that exogenous administration of IL-1 enhances experimental bone/bone-marrow metastases, and that this phenomenon is mediated through prostaglandins. However, neither the constitutive production of IL-1 nor that of prostaglandins appear to play a role in the pathogenesis of bone/bone-marrow metastasis in our murine model system.

Effects of indomethacin, triamcinolone, and dexamethasone on recombinant human interleukin-1-induced substance P and prostaglandin E2 levels in rabbit knee joints

Intra-articular (i.a.) injection of recombinant human interleukin-1 alpha (rHuIL-1 alpha) in rabbit knees induced both an inflammation, as determined by increases in leukocytes in the joint fluid, and cartilage degradation, as measured by loss of proteoglycan. Substance P (SP) and prostaglandin E2 (PGE2) levels in the joint lavage are also elevated. Treatment with 5 mg indomethacin/kg, p.o., b.i.d., 2 mg triamcinolone i.a., and 10 mg dexamethasone/kg, p.o., reduced synovial lavage leukocyte counts, as well as PGE2 and SP lavage concentrations induced with IL-1 injection. However, none of the treatments inhibited rHuIL-1-induced proteoglycan loss.

Immunoregulatory functions of paf-acether. IV. Enhancement of IL-1 production by muramyl dipeptide-stimulated monocytes.

paf-Acether (paf) is a phospholipid mediator of inflammation released from monocytes along with IL-1. In this study, we have examined the role of paf on IL-1 production by human monocytes. When paf from 1 nM to 5 microM, but not its precursor lyso paf, was added to monocytes in the presence of muramyl dipeptide (MDP) or LPS, a marked increase in IL-1 activity over the value with MDP alone was observed. In contrast, paf alone had minimal activity over the same dose range. Antibodies against rHu IL-1 alpha and rHu IL-1 beta neutralized the increased IL-1 activity. Interestingly, MDP that prompts monocytes to synthesize IL-1, induced the synthesis of paf, as well. Most of the paf produced remained cell-associated and always preceded IL-1 synthesis. When the paf receptor antagonist, L-652,731 was added to monocytes, it prevented the enhancement of IL-1 activity induced by exogenous paf. In contrast, L-652,731 had little effect on MDP-induced IL-1 synthesis in the absence of exogenous paf. This may indicate that there are alternative mechanisms involved in the sequences of events leading to IL-1 production. It is also conceivable that the paf receptor antagonist is not able to compete or inhibit endogenous paf as well as it does for exogenous paf. Nevertheless, exogenous paf in association with a second signal, modulates IL-1 production from human monocytes in a positive manner. This may constitute another means through which paf can modulate inflammatory and immune reactions.

Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells.

Two recently identified and purified murine macrophage inflammatory proteins MIP-1 and MIP-2 were tested in vitro both alone, and in combination with purified recombinant (r) murine (mu) GM-CSF, natural (n)muCSF-1, or rhuman (hu)G-CSF, for effects on mouse marrow CFU-GM, in combination with erythropoietin for effects on mouse marrow BFU-E, and in combination with rhuGM-CSF or rhuG-CSF for effects on human marrow CFU-GM. MIP-1 and MIP-2 did not stimulate, but did enhance by up to threefold, colony formation of mouse CFU-GM co-stimulated by rmuGM-CSF and nmuCSF-1, but not by rhuG-CSF, in the absence or presence of serum. MIP-1 and MIP-2 were maximally active at concentrations greater than or equal to 100 ng/ml and the actions appeared to be initiated during the DNA synthetic portion of the cell cycle. Neither MIP-1 nor MIP-2 at up to 1 microgram/ml had any effect on mouse BFU-E, in the absence or presence of erythropoietin. Both MIP-1 and MIP-2 had direct acting effects on purified mouse CFU-GM. The cloning efficiency of 200 purified cells plated with 50 U muCSF-1 was 82% with and 43% without MIP; the cloning efficiency with 50 U rmuGM-CSF was 65% with and 35% without MIP. MIP effects were not mimicked by bacterial LPS, rhuIL-1 alpha, rhuIL-6, or rmuIL-4, and were neutralized by their respective specific antibodies. MIP-1 and MIP-2 also enhanced endogenously stimulated and rhuGM-CSF-, but not rhuG-CSF-, stimulated colony formation by human marrow CFU-GM. These results demonstrate a new role for MIP-1 and MIP-2 in vitro as myelopoietic enhancing activities for CFU-GM.

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors.

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.

In vitro inhibition of arachidonic acid metabolism by two novel retinoid analogs

Ro 23-6457, (all-E)-3,7-dimethyl-9-[2-(trifluoromethyl)-6-(nonyloxy)phenyl]-2, 4,6,8- nonatetraenoic acid, and Ro 23-2895, (all-E)-9-[2-(nonyloxy)phenyl]-3,7-dimethyl-2,4,6,8-nonatetraen oic acid, are two novel retinoid analogs which exhibit antiinflammatory activity in both the developing and the established rat adjuvant arthritis models [8]. Here we investigated the effect of these two compounds on the production of arachidonic acid (AA) metabolites in two in vitro test systems [i.e., Ca2+ ionophore A23187 (I)-stimulated resident rat peritoneal macrophages (MO) and cytokine-stimulated human dermal fibroblasts (HDF)]. Both compounds, Ro 23-6457 and Ro 23-2895, significantly inhibited the release of 14C-AA metabolites and the production of LTB4, PGE2, and 6-keto-PGF1 alpha in I-stimulated MO, at concentrations of 1-33 microM. Both compounds also inhibited the production of PGE2 in HDF stimulated by either rhuIL-1 alpha or huTNF alpha at concentrations of 1 x 10(-5) to 1 x 10(-7) M. Ro 23-2895 was also a potent inhibitor of IL-1-induced collagenase production in rheumatoid synovial cells (IC50 approximately 1 to 2.5 x 10(-8) M). The inhibitory profile of these novel compounds in these cell systems is therefore similar to that of other known antiinflammatory retinoids (e.g., all-trans- and 13-cis-retinoic acid). Inhibitory effects such as those described here might in part contribute to the antiinflammatory activity of these compounds in vivo.

Some recombinant human cytokines stimulate glycosaminoglycan synthesis in human synovial fibroblast cultures and inhibit it in human articular cartilage cultures

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and recombinant human tumor necrosis factor alpha (rHuTNF alpha) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human gamma-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-1 beta was 0.14 pM, while stimulation for rHuIL-1 alpha and rHuTNF alpha was 1.6 pM and 32 pM, respectively. Indomethacin (10 micrograms/ml) had no influence on HA stimulation by cytokines, while hydrocortisone (2-10 micrograms/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of sulfated GAGs. The activity of rHuIL-1 beta was greater than that of rHuIL-1 alpha (half-maximal inhibition at 0.71 pM and 4.7 pM, respectively) and both were considerably more active than rHuTNF alpha; gamma-interferon again had no significant effect. Neither indomethacin nor hydrocortisone influenced cytokine-induced inhibition by either rHuIL-1 preparation. These studies indicate that cytokines released during an inflammatory process may affect GAG synthesis in human joint tissues and may have opposite effects on GAG synthesis in different types of connective tissues.

Modulation of urokinase-type plasminogen activator messenger RNA levels in human synovial fibroblasts by interleukin-1, retinoic acid, and a glucocorticoid.

Previous studies have shown that mononuclear cell-conditioned medium (MCCM), interleukin-1 (IL-1), and all-trans-retinoic acid rapidly stimulate, while glucocorticoids lower, the urokinase-type plasminogen activator (u-PA) activity of human synovial fibroblast-like cells. It is now reported that MCCM, recombinant human IL-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and all-trans-retinoic acid elevate the u-PA messenger RNA (mRNA) levels to a steady-state value within 2 hours, while dexamethasone (10(-7)M) inhibits this increase. For both situations, when the u-PA activity is either stimulated or reduced, the changes in the u-PA mRNA levels parallel the changes in the u-PA activity, and it is suggested that modulation of gene transcription plays an important role.

Antitumor effect of recombinant human interleukin 1 alpha against murine syngeneic tumors.

Interleukin 1 (IL1) has been suggested to have antitumor activity but there is no clear-cut evidence of an in vivo antitumor effect of pure IL1. In this work, the antitumor effect of purified recombinant human IL1 alpha (rHu-IL1 alpha) was assessed on murine tumors transplanted intradermally in syngeneic female mice. rHu-IL1 alpha inhibited the growth of Meth A sarcoma in BALB/c mice, B16 melanoma in C57BL/6 mice and colon 26 adenocarcinoma in CDF1 mice, as well as the spontaneous pulmonary metastasis of Lewis lung carcinoma in BDF1 mice, at intratumoral (itu), intramuscular (im), and/or intravenous (iv) doses ranging from 1 to 30 micrograms/mouse. The antitumor effect of rHu-IL1 alpha was generally dose- and route-dependent, being highest by the itu route, followed by the im and iv routes in that order. Palpable 7-day-old Meth A sarcoma was completely regressed in some mice given rHu-IL1 alpha itu once at doses of 10-30 micrograms/mouse (cured ratio; 71-100%), once a day for 3 days at doses of 3-30 micrograms/mouse (57-86%) or once a day for 7 days at doses of 1-10 micrograms/mouse (14-100%). Palpable 7-day-old B16 melanoma was also regressed completely in some mice given seven itu doses of 10-30 micrograms/mouse (14-86%). One-day-old Meth A sarcoma was more sensitive to rHu-IL1 alpha than 7-day-old Meth A sarcoma. There was no macroscopic sign of inflammation at the site of injection of rHu-IL1 alpha. These results show that rHu-IL1 alpha has antitumor activity in vivo and is worthy of further study as a potential antitumor agent.