Rh Interleukin Research Articles

Chemotherapy-induced severe thrombocytopenia in gynecologic oncology and its treatment with interleukin-3

Six patients suffering from various gynecologic malignancies developed severe thrombocytopenia (WHO IV) after normal-dose chemotherapy. All patients were treated with platelet transfusions but the results on hematopoietic recovery were not satisfactory. Therefore treatment with a new hematopoietic substance (rh interleukin-3) was initiated. All patients received a dosage of 5 mu g/kg/bw daily s.c. up to 10 days depending on the response of platelet counts. In two women, who had suffered from severe hemorrhage (epistaxis resp. ankle joint hematoma), symptoms disappeared after approximately three days of rhIL-3 use. Whereas platelet transfusions were ineffective in all patients, IL-3 led to a significant increase of the platelet count after 3-5 days of application. Side-effects were mild, when seen in one case, where G-CSF was given at the same time. Our experience supports the idea of using new growth factors like rhIL-3 to cure chemotherapy-induced myelosuppression, such as severe thrombocytopenia.

Effects of thrombopoietin (c-mpl ligand) on growth of blast cells from patients with transient abnormal myelopoiesis and acute myeloblastic leukemia.

Thrombopoietin (TPO) is a ligand for c-mpl that promotes both proliferation and differentiation of megakaryocytes in vivo and in vitro. We investigated the expression of c-mpl transcripts and the effects of recombinant human TPO (rhTPO) on the proliferation and differentiation of human leukemic cell lines or fresh samples obtained from 32 patients with transient abnormal myelopoiesis (TAM) or acute myeloblastic leukemia (AML). Cells were cultured with TPO alone or combined with rh interleukin-3 (IL-3) or stem cell factor (SCF). Expression of c-mpl was verified in 6 of 13 cases tested. All but one of the cases that showed c-mpl expression responded to TPO. Blasts from all cases of TAM or French-American-British (FAB) subtype M7 showed growth responses to TPO with higher sensitivity than cells of other FAB subtypes and these responses were increased by addition of rhIL-3 or rhSCF in some cases. Responses of cells of other FAB subtypes varied. In addition, increased expression of platelet-specific surface antigens on MO7E cells after incubation with rhTPO was observed. These data suggest that TPO may be involved in the abnormal proliferation and differentiation of human leukemic cells, especially of M7 and TAM cells, considered to be of megakaryocytic lineage.

Clinical and immunomodulatory effects of repetitive 2-day cycles of high-dose continuous infusion IL-2.

High-dose interleukin-2 (IL-2) treatment has demonstrated promising antitumour activity in renal cell carcinoma (RCC) and malignant melanoma (MM) and has been shown to induce broad immunological effects. The optimal IL-2 dose and schedule, however, still remain to be defined. We studied a treatment protocol consisting of five repetitive cycles of high-dose recombinant (rh) IL-2 (24 x 10(6) U/m2/day) administered weekly on two consecutive days by continuous intravenous infusion. 17/19 were RCC patients, 2 of whom responded with a complete remission (CR) and 3 with a partial response (PR) (CR + PR: 29%; median response duration of 11.5+ months (range: 3-14 months)). IL-2 induced a pronounced increase of lymphocytes and pro-inflammatory cytokines IL-8, IL-5, gamma-IFN, TNF- alpha and TNF-beta (p < 0.05) that peaked in cycle 3. With subsequent therapy, serum levels of these cytokines, NK, T cells and eosinophils decreased, whereas serum IL-10 levels progressively increased with maximum levels achieved after the fifth week of treatment, suggesting that it may be involved in dampening the inflammatory response induced by IL-2. Absolute numbers of activated T cells and NK cells remained elevated as compared to baseline for at least 4 weeks after treatment cessation. Based on these observations, future scheduling of IL-2 will be done at 3 weekly 2-day cycles separated by a week 4 treatment-free interval in order to increase further the 29% objective response rate achieved in this study.

Nicardipine hydrochloride suppresses DNA synthesis in human mesangial cells stimulated with recombinant human (rh) platelet-derived growth factor AA, rh interleukin-1 alpha, or rh tumor necrosis factor-alpha.

In order to define the role of cytokines in mesangial cell pathophysiology, we measured the mitogenic activity of recombinant human platelet-derived growth factor AA (rhPDGF-AA), rh interleukin-1 alpha (rhIL-1 alpha) and rh tumor necrosis factor-alpha (rhTNF-alpha) in cultured human mesangial cells, and investigated the effect of the calcium channel blocker nicardipine hydrochloride on their cell mitogenic activity. DNA synthesis in mesangial cells, stimulated by rhPDGF-AA, rhIL-1 alpha or rhTNF-alpha, was measured using [3H]TdR up take and similar investigations, with nicardipine hydrochloride added to the above, were conducted. The results showed DNA synthesis in cultured human mesangial cells were stimulated by rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha, and this effect was reduced by the addition of nicardipine hydrochloride. Since rhPDGF-AA, rhIL-1 alpha and rhTNF-alpha are released by the inflammatory cells which infiltrate glomeruli, these cytokines may be involved in the mechanisms of mesangial cell proliferation observed in immune-mediated mesangial proliferative glomerulonephritis. Nicardipine hydrochloride reduced DNA synthesis in cultured human mesangial cells in response to these cytokines, suggesting possible application in medical therapy for immune-mediated mesangial proliferative glomerulonephritis.

Effect of interleukin-1 β, tumour necrosis factor-α and interleukin-6 on the control of thyrotropin secretion

Since serum concentrations of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) are elevated in infectious and inflammatory illnesses, we examined their potential role in contributing to the low TSH concentrations associated with such conditions, both at the level of the pituitary and the hypothalamus. 20 hours exposure to recombinant murine TNF-a (10 −11 to 10 −10mol/l) enhanced the basal and the TRH-stimulated release of TSH by cultured rat anterior pituitary cells, but 4 hours exposure increased only basal TSH secretion. Recombinant human (rh) IL-1β, at a dose of 10 −11mol/1 only, produced a very small increase in basal TSH secretion after 4h, but not 20h, exposure. TRH-stimulated TSH secretion was not affected by IL-1β in concentrations up to 10 −10 mol/1, at either exposure time. Rh IL-6 (10 −12 to 10 −9 mol/1), had no effect on basal or TRH-stimulated TSH secretion at either exposure time. TNF-α, IL-lβ, and IL-6 all failed to modify the inhibitory response to triiodothyronine (T 3) and thyroxine (T 4) on TSH secretion, under basal or TRH-stimulated conditions. Indirect effects of the cytokines on the stimulation or inhibition of TSH secretion, via TRH or SRIF respectively, were tested in isolated rat hypothalamic slices. 30 min exposure to TNF-α, IL-1β, or IL-6 had no effect on the basal release of SRIF. However, IL-1β, from 2.5x10 −12to 10 −10 mol/l, produced a dose-dependent enhancement of the SRIF released by 5×10 −12mol/l extracellular K +. The effect appeared to be mediated via IL-1 receptors, and to involve prostanoid formation, since it was inhibited by IL-1 receptor antagonist protein, 10 −7 mol/1, and indomethacin, 2.8×10 −5mol/l, respectively. Neither basal nor K +-stimulated TRH release was influenced by TNF-α, IL-1β, or IL-6. The results indicate that direct effects of these cytokines on the pituitary do not contribute to reduced circulating TSH concentrations during inflammation and infection, but that enhanced hypothalamic release of SRIF, in response to elevated IL-1β, could contribute to such a decrease in TSH. None of the cytokines tested decreased hypothalamic TRH release in vitro. However, further in vivo experiments would be required to determine whether a longer exposure to these agents could reduce TRH release either directly, or indirectly via inputs from outside the hypothalamus.

Differential response of human basophils and mast cells to recombinant chemokines

Chemokines are proinflammatory peptides regulating the functions of various hematopoietic cells. We have analyzed the effects of seven recombinant human (rh) chemokines (MCAF, RANTES, MIP-1 alpha, MIP-1 beta, IL-8, GRO, and IP-10) on the growth and function of human basophils and mast cells. We found that MCAF, but not RANTES, MIP-1 alpha, MIP-1 beta, IL-8, GRO, or IP-10, causes direct and dose-dependent histamine release from basophils (MCAF, 5 micrograms/ml: 26.9 +/- 3.4%; other chemokines: < 5% of total histamine). An increased (2.1 to 3.5-fold) response to MCAF was obtained when basophils were preincubated with rh interleukin-3 (100 units/ml). Moreover, IL-3-primed basophils became responsive to physiologic concentrations (< 1 microgram/ml) of MCAF, IL-8, and RANTES. None of the chemokines tested was able to induce histamine secretion in mast cells obtained from lung (n = 2), skin (n = 1), uterus (n = 3), or tonsils (n = 3), even when cells had been preincubated with the mast cell agonist SCF. The chemokines also failed to modulate the expression of activation antigens (CD11b/C3biR, CD25/IL-2R beta, CD63, IL-3R alpha, CD117/c-kit) on the mast cell line HMC-1 or the basophil cell line KU-812 and were unable to induce differentiation of basophils or mast cells in culture. Together, our results show that basophils respond to rhIL-8, rhMCAF, and rhRANTES and that, unlike human basophils, human mast cells are unresponsive to recombinant chemokines.

Myelosuppression in HCL: Role of hairy cells, T cells and haematopoietic growth factors

To elucidate mechanisms which may be responsible for the haematopoietic insufficiency in hairy cell leukaemia (HCL), we investigated in an autologous in vitro system the influence of haematopoietic growth factors (CSFs) and the effects of hairy cells (HCs) as well as T cells on the formation of haematopoietic colonies (CFU). Colony forming assays were performed using peripheral blood mononuclear cells (PBMC) of 6 HCL patients. To remove HCs, PBMCs were subjected to complement-mediated lysis, T cells were removed by E-rosette formation. Assays were done with and without recombinant human (rh) interleukin-3 (IL-3) and rh granulocyte-macrophage-colony-stimulating factor (GM-CSF). All 6 patients exhibited a severe reduction of their circulating progenitor cell (CPC) compartment. There was no correlation between the degree of colony reduction and the number of HCs. However, a correlation was found between the numbers of CPCs of HCL patients and healthy donors and the monocyte counts in these groups (r = 0.8573, p < 0.001). The removal of autologous HCs, but also of T cells, resulted in a significant increase in colony formation (BFU-E, CFU-GM, CFU-mix). In none of the experiments, however, did colony numbers come close to the normal range. This was only achieved by supplementation of the culture medium with rh IL-3 and rh GM-CSF. The results suggest that the haematopoietic failure observed in HCL patients is probably due to an inadequate supply of CSFs as well as to an inhibitory activity of HCs and T cells which might exert their effects in a synergistic fashion. There is also evidence that the lack of monocytes plays a role in the development of the haematopoietic insufficiency in HCL.

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.

Preclinical analysis of cytokine therapy in the SCID-hu mouse

A severe combined immunodeficient (SCID)-hu mouse model implanted with human fetal bone was used to assess the effects of various recombinant human (rh) hematopoietic growth factors, administered either alone or in combination, on human hematopoiesis in vivo. Treatment with rh granulocyte colony-stimulating factor (G-CSF) elicited the expansion of mature neutrophilic granulocyte populations in human marrow. Administration of rh interleukin-3 (IL-3) induced significant increases of eosinophilic granulocyte and burst-forming unit, erythrocyte (BFU-E) activity. The rhIL-6 did not cause significant changes in the subpopulations of human hematopoietic cells within the grafts, but did increase the number of colony-forming unit, granulocyte-macrophage and BFU-E. Pretreatment with rhIL-3 followed by rh erythropoietin (Epo) administration enhanced Epo-induced human erythropoiesis significantly. No synergistic effects on myelopoiesis were observed using sequential treatment with rhIL-3 followed by rhG-CSF. Instead, these factors seemed to work independently, with rhG-CSF increasing the percentage of neutrophils and rhIL-3 increasing the percentage of eosinophils. When administered simultaneously with rhEpo, rhIL-6 showed dose-dependent inhibitory effects on in vivo Epo-induced human erythropoiesis. The rhIL-6 also caused a reduction in the percentage of human neutrophils induced by rhG-CSF. These results suggest that the SCID-hu mouse provides a useful small animal model to assess the in vivo effects of hematopoietic growth factors on human hematopoiesis.

Preclinical Analysis of Cytokine Therapy in the SCID-hu Mouse

AbstractA severe combined immunodeficient (SCID)-hu mouse model implanted with human fetal bone was used to assess the effects of various recombinant human (rh) hematopoietic growth factors, administered either alone or in combination, on human hematopoiesis in vivo. Treatment with rh granulocyte colony-stimulating factor (G-CSF) elicited the expansion of mature neutrophilic granulocyte populations in human marrow. Administration of rh interleukin-3 (IL-3) induced significant increases of eosinophilic granulocyte and burst-forming unit, erythrocyte (BFU-E) activity. The rhIL-6 did not cause significant changes in the subpopulations of human hematopoietic cells within the grafts, but did increase the number of colony-forming unit, granulocyte-macrophage and BFU-E. Pretreatment with rhIL-3 followed by rh erythropoietin (Epo) administration enhanced Epo-induced human erythropoiesis significantly. No synergistic effects on myelopoiesis were observed using sequential treatment with rhIL-3 followed by rhG-CSF. Instead, these factors seemed to work independently, with rhG-CSF increasing the percentage of neutrophils and rhIL-3 increasing the percentage of eosinophils. When administered simultaneously with rhEpo, rhIL-6 showed dose-dependent inhibitory effects on in vivo Epo-induced human erythropoiesis. The rhIL-6 also caused a reduction in the percentage of human neutrophils induced by rhG-CSF. These results suggest that the SCID-hu mouse provides a useful small animal model to assess the in vivo effects of hematopoietic growth factors on human hematopoiesis.

Fatal spleen rupture during induction chemotherapy with RH GM-CSF priming for acute monocytic leukemia. Clinical case report and in vitro studies

Recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-SCF) is currently being tested in clinical trials for the treatment of acute myeloid leukemias with two main intentions: reduction of neutropenia and recruitment of leukemic blasts into cell cycle to enhance cytarabine (ara-C) mediated cytotoxicity. We report a case of a fatal spleen rupture in a patient with acute monocytic leukemia (AML M5b) who was treated according to a clinical phase I/II protocol with rh GM-CSF priming and standard induction chemotherapy TAD 9 (thioguanine/ara-C/daunorubicin). During treatment we observed rapidly rising peripheral blast counts and the development of an acute abdomen. Ultrasound examination revealed splenomegaly due to diffuse cellular infiltration and spleen rupture. The patient died 17 days later due to pneumonia and renewed spleen hemorrhage. Bone marrow progenitor assays before treatment showed exclusive growth of monocytoid blast cell colonies (CFU-L). Colony growth could be stimulated with rh GM-CSF and blocked dose-dependently by a monoclonal anti-GM-CSF antibody. CFU-L proliferation also increased after stimulation with rh interleukin-3 (rh IL-3) and supra-additively with rh granulocyte colony-stimulating factor (rh G-CSF) combined with rh GM-CSF. Furthermore, rh GM-CSF induced surface marker expression of CDw 65 and CD 11b on isolated CFU-L blasts. After short-term suspension culture, rh GM-CSF enhanced the expression of CD 29- and CD 11b-adhesion molecules on peripheral blast cells. In summary, this case represents a fatal spleen rupture occurring during rh GM-CSF priming and induction chemotherapy for acute monocytic leukemia. Although the etiology of this spleen rupture remains uncertain, in view of our data we suggest special caution, when further testing this therapy protocol in acute leukemias with monocytic subtype and high peripheral blast cell counts.

Efficacy of Low Doses of the Polyethylene Glycol Derivative of Interleukin-2 in Modulating the Immune Response of Patients with Human Immunodeficiency Virus Type 1 Infection

Interleukin-2 (IL-2) is a key cytokine in cellular immunity. Human immunodeficiency virus type 1 (HIV-1)-infected individuals lack IL-2 because of low CD4+ T lymphocyte numbers. In an attempt to enhance cellular immunity, low-dose recombinant human (rh) IL-2 at 10 micrograms or 180,000 units or its polyethylene glycol (PEG) derivative at 9 micrograms or 36,000 units was given by intracutaneous injection to 8 HIV-1-infected men for 30 days. Participants had no evidence of opportunistic infection and received concurrent zidovudine. IL-2 treatment was nontoxic and elicited a local cellular response resembling classic delayed-type hypersensitivity (DTH) with local interferon-gamma production, even in anergic patients. Systemic responses included enhanced DTH responses to recall antigens, improved in vitro proliferative responses to mitogen, and enhanced NK cell activity. Peripheral leukocyte phenotype and virus titers were unchanged. Long-term studies of low-dose IL-2 are warranted to determine whether immunoenhancing effects can be sustained and if they are associated with improved clinical course.

Expression of functional receptors for interleukin-6 by human polymorphonuclear, leukocytes Downregulation by granulocyte-macrophage colony-stimulating factor

Surface interleukin-6 receptors were identified on human polymorphonuclear leukocytes (PMNL) by monoclonal anti p80-chain antibody MT 18 Cytoplasmic RNA harvested from PMNL also contained IL-6-R transcripts. Binding of recombinant human (rh) interleukin-6 (IL-6) to IL-6-R bearing PMNL was identified by flow cytometry using phycocrythrin (PE)-conjugated ligand. Treatment of PMNL with rh granulocyte-macrophage colony-stimulating factor (GM-CSF) led to the inability of PMNL to bind MT 18 monoclonal antibody (moAb) and to display binding sites for PE-conjugated rh IL-6. Levels of IL-6-R transcripts in PMNL exposed to GM-CSF were about 5-fold below those of PMNL cultured in medium only. Though a definitive role for IL-6 to modulate the function of PMNL was not found, treatment of PMNL with rh IL-6 clearly resulted in an enhancement of transcript levels of the early response genes c-fox and c-jun in these cells, thus indicating that IL-6 binding is followed by signal transduction.

Neuromodulation of fever: Apparent involvement of opioids

It was recently reported that the opiate antagonist, naloxone (Nal), blocks the changes induced by the endogenous pyrogen, interferon- α 2 (IFN), in the electrical activity of hypothalamic thermosensitive neurons in rat brain slice preparations. This study was undertaken to determine whether the pyrogenic response to this cytokine might, therefore, be modulated through Nal-reversible opiate receptors. To examine this possibility, conscious guinea pigs were injected IV with recombinant human (rh) IFN (10 MU/animal), or, for comparison, with S. enteritidis endotoxin (lipopolysaccharide, LPS; 2 μg/kg), rh tumor necrosis factor-α (TNF; 20 μg/kg), or rh interleukin-6 (IL6; 50 μg/kg); Nal (10 mg/kg, SC) was administered immediately before the pyrogens. And also for comparison, in separate experiments, indomethacin (Indo; 10 mg/kg, IM) was injected 20 min before the pyrogens. Both Nal and Indo abolished the febrile rises evoked by IFN, TNF, and IL6. Nal reduced the first and suppressed the second of the characteristically bimodal febrile response to LPS; Indo depressed both peaks. Neither blocker had any significant thermal effect by itself. These results suggest that two processes may mediate the pyrogenic effects of these substances, viz., an endogenous opioid- and a PGE-dependent mechanism.

Identification of structural differences between different forms of interleukin-2 (IL-2) using anti-(human recombinant) IL-2 monoclonal antibodies

We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (Kaff approximately equal to 10(-8) M) and are of the IgG1 isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh IL-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IL-2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID50, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 micrograms/ml to 30 micrograms/ml final concentrations of the antibodies. Using different forms of IL-2 we found that the mAbs give different patterns of inhibition of the IL-2-dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2.

Interleukin-6 (IL-6) is an intermediate in IL-1-induced proliferation of leukemic human megakaryoblasts

We have examined the in vitro effects of recombinant human (rh) interleukin-1 (IL-1) on the growth of purified megakaryoblasts obtained from patients with acute megakaryoblastic leukemia. We demonstrate that both IL-1 alpha and IL-1 beta treatment of these cells led to stimulation of DNA synthesis (as shown by increase of 3H-thymidine incorporation up to 35-fold) and also resulted in colony formation of leukemic megakaryoblasts. However, the stimulatory effect of IL-1 was dependent on endogenous production of IL-6, because addition of neutralizing monoclonal antibody (MoAb) to IL-6 abrogated the stimulatory activity of IL-1. In contrast, neutralizing MoAbs to granulocyte (G)-colony stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF failed to counteract the growth- enhancing effects of IL-1. Leukemic megakaryoblasts accumulated IL-6 mRNA and released IL-6 protein into their culture supernatant when exposed to rh IL-1 but failed to disclose transcripts for G-, GM-, and M-CSF under these conditions. Analysis of IL-6 receptor (IL-6R) transcript levels demonstrated that megakaryoblasts constitutively expressed IL-6R mRNA and that these transcripts are down-regulated to undetectable levels upon exposure to IL-1 and IL-6. Increase of 3H- thymidine incorporation by megakaryoblasts could be duplicated by exogenous IL-6 that could be blocked by neutralizing MoAb to IL-6. In conclusion, our results suggest that leukemic megakaryoblasts could produce and secrete IL-6, and express IL-6R, and that the growth- enhancing effect of IL-1 on these cells is indirect, via production of IL-6 by leukemic cells.

Interleukin-6 (IL-6) Is an Intermediate in IL-1–Induced Proliferation of Leukemic Human Megakaryoblasts

We have examined the in vitro effects of recombinant human (rh) interleukin-1 (IL-1) on the growth of purified megakaryoblasts obtained from patients with acute megakaryoblastic leukemia. We demonstrate that both IL-1 alpha and IL-1 beta treatment of these cells led to stimulation of DNA synthesis (as shown by increase of 3H-thymidine incorporation up to 35-fold) and also resulted in colony formation of leukemic megakaryoblasts. However, the stimulatory effect of IL-1 was dependent on endogenous production of IL-6, because addition of neutralizing monoclonal antibody (MoAb) to IL-6 abrogated the stimulatory activity of IL-1. In contrast, neutralizing MoAbs to granulocyte (G)-colony stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF failed to counteract the growth-enhancing effects of IL-1. Leukemic megakaryoblasts accumulated IL-6 mRNA and released IL-6 protein into their culture supernatant when exposed to rh IL-1 but failed to disclose transcripts for G-, GM-, and M-CSF under these conditions. Analysis of IL-6 receptor (IL-6R) transcript levels demonstrated that megakaryoblasts constitutively expressed IL-6R mRNA and that these transcripts are down-regulated to undetectable levels upon exposure to IL-1 and IL-6. Increase of 3H-thymidine incorporation by megakaryoblasts could be duplicated by exogenous IL-6 that could be blocked by neutralizing MoAb to IL-6. In conclusion, our results suggest that leukemic megakaryoblasts could produce and secrete IL-6, and express IL-6R, and that the growth-enhancing effect of IL-1 on these cells is indirect, via production of IL-6 by leukemic cells.

Vascular Cell Adhesion Molecule-1 Mediates Lymphocyte Adherence to Cytokine-Activated Cultured Human Endothelial Cells

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells [published erratum appears in Blood 1990 Dec 1;76(11):2420

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF- activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

The action of bryostatin on normal human hematopoietic progenitors is mediated by accessory cell release of growth factors

Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34- positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony- stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone- marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.