Spontaneous and interleukin 2 induced secretion of tumour necrosis factor and gamma interferon following autologous marrow transplantation or chemotherapy

Culture of lymphocytes from allograft recipients or from normal donors with Interleukin 2 (IL2) induces high levels of gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) secretion. We now show that production of these two cytokines can also be increased after autologous bone marrow transplantation (BMT) and induced following chemotherapy for haematological malignancy. These effects occur even though the regenerating IL2 responsive lymphocytes in this context have previously been extensively exposed to cytotoxic agents. IL2 induced secretion of gamma-IFN and TNF is higher in autograft recipients than in patients treated by chemotherapy alone. This effect may be related to differences in the phenotypic profile between recovering lymphocytes in the two groups and to an increased degree of prior in vivo lymphocyte activation in the autologous bone marrow transplant recipients. In both groups of patients, IL2 acts on CD3+ T cells and on CD16+ NK cells so that depletion of either subset incompletely abrogates IL2 dependent gamma-IFN secretion. As both TNF and gamma-IFN possess anti-leukaemic and anti-infective activity, enhancement of their secretion by IL2 infusion after autologous bone marrow transplant and induction after chemotherapy may be of therapeutic benefit.     

Interleukin 2 infusion induces haemopoietic growth factors and modifies marrow regeneration after chemotherapy or autologous marrow transplantation

Administration of interleukin 2 (IL2) to patients with minimal residual malignant disease following myeloablative chemo-radiotherapy may augment immune reconstitution and reduce the risk of relapse by increasing cytotoxic effector function and cytokine dependent killing directed at residual malignant cells. The ability of IL2 generated activated killer cells to inhibit haemopoietic progenitor cells and to release gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) may, however, retard haemopoietic recovery, as both TNF and gamma IFN inhibit normal myelopoiesis in vitro. To determine the effect of IL2 infusion on myeloid regeneration in vivo, we have examined haemopoietic recovery in patients receiving this cytokine following autologous marrow transplantation or ablative chemotherapy. We find that IL2 infusion accelerates neutrophil recovery and that granulocyte-macrophage colony stimulating factor (GMCSF) and IL3 mRNA become detectable in circulating mononuclear cells. Induction of TNF by IL2 may also contribute to subsequent acceleration of myelopoiesis by initiation of GM-CSF mRNA synthesis in patient marrow fibroblasts. These results show that IL2 infusion may facilitate myeloid recovery when administered during the period of haemopoietic regeneration following ablative chemoradiotherapy.     

Acute hematologic effects of interferon alpha,interferon gamma,tumor necrosis factor alpha and Interleukin 2

Summary This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFN-gamma Interleukin 2 and tumor necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFN-gamma also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.     

Serum interleukin-3 levels following autologous or allogeneic bone marrow transplantation: effects of T-cell depletion, blood stem cell infusion, and hematopoietic growth factor treatment

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme- linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL- 3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.     

Recombinant interleukin-2 and interferon alpha immunotherapy following autologous bone marrow transplantation. A case report of cardiovascular toxicity with serial echocardiographic evaluation.

The most serious side effects of recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN alpha) immunotherapy are cardiovascular disturbances, including systemic hypotension, left-ventricular dysfunction and pulmonary edema. We present a 25-year-old female who developed reversible cardiogenic shock during intermediate dose rIL-2 and low dose rIFN alpha therapy. Rapid clinical improvement occurred after intravenous fluid and dopamine support. A serial echocardiographic evaluation, which has not been described previously in this setting, is reported.     

Inflammatory peripheral neuropathy following high dose chemotherapy and autologous bone marrow transplantation.

A 40-year-old man with non-Hodgkin's lymphoma developed severe ascending sensorimotor neuropathy 10 days after treatment with high dose chemotherapy and autologous bone marrow rescue. The neuropathy had axonal plus demyelinating features on electrophysiological studies. Sural nerve biopsy showed heavy infiltration of the epineurium and endoneurium with mononuclear cells. The patient had no other evidence of graft-versus-host disease. He failed to respond to plasmapheresis but responded to high dose steroids.     

Myelodysplasia and acute leukemia following high-dose chemotherapy and autologous bone marrow or peripheral blood stem cell transplantation.

Therapy-related myelodysplastic syndrome (t-MDS)/acute myeloid leukemia (t-AML) has been reported after autologous bone marrow or peripheral blood stem cell transplantation (ABMT/PBSCT) for various malignancies. We retrospectively reviewed all adult ABMT/PBSCT cases performed at the University of Chicago Medical Center from 1985 to 1997 in order to determine the incidence of therapy-related leukemia. Among 649 patients, seven (1.1%) developed therapy-related acute lymphoblastic leukemia (one patient) or t-MDS/t-AML (six patients). Of these seven, primary malignancies included one case of breast carcinoma, five cases of Hodgkin's disease (HD) and one case of non-Hodgkin's lymphoma (NHL). Disease-specific incidences for therapy-related leukemia occurring after ABMT/PBSCT were one in 354 (0.3%) for breast carcinoma, five in 79 (6.3%) for HD and one in 103 (1%) for NHL. The median latency periods for the development of therapy-related leukemia from the time of initial diagnosis and of ABMT/PBSCT were 5.5 and 1.5 years, respectively, for the combined HD and NHL group of patients and 4.4 and 2.8 years, respectively, for the one breast carcinoma patient. All seven patients had clonal cytogenetic abnormalities, and five had recurring abnormalities typical of myeloid disorders. Given the similar latency period observed in patients treated with conventional chemotherapy alone, our findings support the hypothesis that therapy-related leukemia after ABMT/PBSCT likely results from pre-transplant therapy. Early detection of therapy-related leukemia is therefore critical to exclude these patients from undergoing ABMT/PBSCT.     

Inhibition of human granulocyte-macrophage colony formation by interleukin 2-treated lymphocytes is mediated by interferon gamma and tumor necrosis factor alpha

We previously demonstrated that human granulocyte-macrophage colony (granulocyte-macrophage colony-forming units, CFU-GM) formation was inhibited by interleukin 2 (IL-2)-treated lymphocytes and their conditioned medium (CM). In the present study, the mechanism of this suppression was investigated. When anti-interferon (IFN)-gamma antibody or anti-tumor necrosis factor (TNF)-alpha antibody was added to CFU-GM agar culture with IL-2-treated lymphocytes or their CM, the inhibition of CFU-GM colony formation was partially abrogated, whereas anti-TNF-beta antibody did not abolish the inhibitory effects. When anti-IFN-gamma and anti-TNF-alpha antibodies were added simultaneously, full recovery of colony formation was observed. In the CM of IL-2-treated lymphocytes, detectable levels of IFN-gamma (81 +/- 15 U/ml) and TNF (3.1 +/- 1.1 U/ml) were found. Addition of IFN-gamma and TNF-alpha at these concentrations to the agar culture inhibited CFU-GM colony formation. Taken together, these results indicate that inhibition of human CFU-GM colony formation by IL-2-treated lymphocytes and their CM is mediated by IFN-gamma and TNF-alpha generated from IL-2-treated lymphocytes.     

Hypophysectomy inhibits the synthesis of tumor necrosis factor alpha by rat macrophages: partial restoration by exogenous growth hormone or interferon gamma

We recently demonstrated that GH and interferon-gamma (IFN gamma) act in a similar manner to prime macrophages in vitro and in vivo for enhanced superoxide anion release. In this report we investigated the physiological role of the pituitary gland and GH in in vivo priming of resident peritoneal macrophages for the synthesis of tumor necrosis factor-alpha (TNF alpha) in vitro. Compared to normal rats, hypophysectomized animals had an 83% reduction in macrophage production of TNF alpha after in vitro stimulation with lipopolysaccharide. Sham operation had no significant effect on the ability of macrophages to secrete TNF alpha in response to lipopolysaccharide. Both native pituitary-derived porcine GH (48 micrograms/rat.9 days) and native pituitary-derived rat GH (96 micrograms/rat.9 days) more than tripled the in vitro production of TNF alpha by macrophages from hypophysectomized rats (342 and 358 vs. 112 U/mg protein for placebo-treated rats, respectively). Each of these preparations of GH also increased growth more than 6-fold in hypophysectomized rats (32 and 30 g vs. 5 g in placebo controls). Heat inactivation of native pituitary-derived porcine GH significantly reduced its in vivo ability to augment both TNF alpha synthesis by macrophages and body growth. Recombinant rat IFN gamma (2000 U/rat.9 days) more than tripled the production of TNF alpha by macrophages from hypophysectomized rats (343 vs. 112 U/mg protein). In contrast to its in vivo effects, addition of GH in vitro to macrophages from hypophysectomized rats did not prime these cells for the synthesis of TNF alpha, indicating an indirect mechanism of action for GH. To further test the biological relevancy of GH with respect to synthesis of TNF alpha, hemorrhagic necrosis of TNF alpha-sensitive murine methyl-cholanthrene-induced tumors was assessed in pituitary-intact mice. Native porcine GH (133 micrograms/mouse.7 days) significantly augmented both the necrosis to tumor ratio and the hemorrhage to tumor ratio. These findings establish the physiological relevance of the pituitary gland and GH in the priming of macrophages for TNF alpha synthesis.     

Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon gamma.

Interleukin (IL)-2, IL-4, IL-7, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma) has been shown to be able to induce tumor rejection if produced locally by the tumor cells after gene transfer. To analyze whether the cellular rejection mechanisms are different or redundant we have expressed the cytokines in the same tumor cell line (J558L). Cell depletion experiments revealed that all cytokines required CD8+ T cells for complete long-term tumor eradication, although effective but transient host-dependent tumor suppression was also observed in the complete absence of CD8+ T cells. The transient tumor suppression induced by IL-2, IL-4, TNF, or IFN-gamma was also operative in nude and severe combined immunodeficient mice, whereas only tumor suppression induced by IL-7 was dependent on the presence of CD4+ T cells and was not evident in nude mice. The T-cell-independent effector arm of IL-2 and IFN-gamma but not IL-4 and TNF was mediated in part by natural killer cells. The transience of tumor suppression in the absence of T cells reflected loss of cytokine production in the case of TNF, IL-2, and IL-4 but not IFN-gamma. Immunohistologic analysis revealed all cytokine-producing tumors to be heavily infiltrated by macrophages. IL-4 and IL-7 tumors additionally contained eosinophils. The infiltration by T cells did not necessarily reflect their contribution to tumor rejection. Thus, the different cytokines activate heterogeneous transient tumor-suppressive mechanisms but always require CD8+ T cells for complete tumor rejection.     

Maintenance treatment with recombinant alpha interferon after autologous bone marrow transplantation for aggressive myeloma in first remission after conventional induction chemotherapy

Twenty patients (median age 57 years) with high tumor mass myeloma in first remission after conventional chemotherapy received a two-phase treatment: autologous bone marrow transplantation (ABMT) using a preparative regimen of high dose melphalan plus total body irradiation followed by maintenance treatment with recombinant alpha interferon. Before ABMT all patients were in partial remission, while after ABMT 10 (50%) achieved complete remission, and 10 remained in partial remission (with a 90% decrease in measurable paraprotein in 7/10). With a median follow-up of 19.8 months (12.2-33.5) after diagnosis and 13 months (4-25) after ABMT, the Kaplan-Meier 2-year post-ABMT probability of progression-free survival was 85% (95% CI = 58.7-95.8%). None of the 10 patients in complete remission has relapsed. No toxic death occurred. Alpha interferon was introduced early after ABMT (2.7 months) and was well tolerated. This strategy may represent an advance in the management of aggressive myeloma.     

Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) can produce prolonged clinical remission in some patients with hematologic malignancies. Unfortunately, disease relapse may occur despite BMT. Studies in animal models and clinical experience have provided evidence that immunologic factors play an important role in preventing relapse post-BMT. To stimulate immunologic activity in patients post-BMT, we administered prolonged uninterrupted continuous infusions of low-dose recombinant interleukin-2 (rIL-2). Thirteen marrow recipients (seven autologous BMT, six CD6 T-depleted allogeneic BMT) received rIL-2 at a dose of 2 x 10(5) U/m2/d for a scheduled period of 90 days. rIL-2 was administered through a Hickman catheter with a portable pump beginning a median of 85 days after BMT. Toxicity was minimal and all treatment could be undertaken in the outpatient setting. No patient developed any signs of graft-versus-host disease, hypotension, or pulmonary capillary leak syndrome. Treatment did not affect the absolute neutrophil count or hemoglobin level, but eosinophils increased substantially in most patients. Platelet counts decreased by 20% in 10 of 13 individuals within 2 weeks, but stabilized thereafter. Despite the low dose of rIL-2 administered, significant immunologic changes were noted. Specifically, all 13 patients experienced a marked increase (fivefold to 40-fold) in natural killer (NK) cell number. Phenotypic characterization showed that the majority of NK cells were CD56bright+ CD16+ CD3-. In contrast, a minor increase in T-cell number was noted in only 4 of 13 patients. Low-dose rIL-2 treatment resulted in augmentation of in vitro cytotoxicity against K562 and COLO tumor targets. This cytotoxic activity could be dramatically enhanced by incubation with additional rIL-2 in vitro. The immunologic effects of rIL-2 treatment were similar in both autologous and allogeneic marrow recipients. Our data suggest that prolonged infusion of rIL-2 at low doses is safe and can selectively increase NK cell number and activity after BMT. Further studies to assess the impact these changes may have on disease relapse post-BMT will be undertaken.     

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells

The cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-gamma) were studied in five human pancreatic tumor cell lines. During a 48-h incubation, MIA PaCa-2 cells were most sensitive to rhTNF (56% cytotoxicity, 500 U/ml), T3M4 cells were most sensitive to rhIFN-gamma (54% cytostasis, 250 U/ml), and ASPC-1 and COLO 357 cells were most sensitive to the combination of rhTNF and rhIFN-gamma (56 and 55% cytotoxicity, respectively, 250 U/ml of each cytokine). The PANC-1 cells were relatively insensitive to either the individual or the combined effects of these cytokines. All five cell lines exhibited specific, high-affinity receptors for 125I-labeled rhTNF (480-8,610 sites/cell) and rhIFN-gamma (2,050-6,280 sites/cell). The MIA PaCa-2 cells, which were the most sensitive to the inhibitory effects of rhTNF, also possessed the largest number of 125I rhTNF receptors; all other cell lines had a relatively low number of binding sites and low sensitivity. In contrast, no direct correlation could be made between the number of IFN-gamma binding sites and inhibitory sensitivity in any of the cell lines. Incubation of COLO 357 cells at 37 degrees C with either 125I rhTNF or 125I rhINF-gamma led to internalization of the respective 125I-labeled ligand. Our findings document the presence of cytokine receptors in human pancreatic carcinoma cells and suggest that postreceptor events rather than differences in receptor number or affinity more likely govern the responsiveness of pancreatic cancer cells to TNF and IFN-gamma.     

Long-term haematological recovery following high-dose chemotherapy with autologous bone marrow transplantation or peripheral stem cell transplantation in patients with solid tumours.

long-term peripheral blood counts and factors influencing long-term trilineage haematological recovery of consecutive patients in a single institution treated with high-dose chemotherapy (hdc) and abmt or psct for solid tumours were examined. patients with a relapse-free survival of >1 year were included in the analysis (n = 131). Peripheral blood counts were examined 6 months and yearly following transplantation. Median follow-up was 4.1 years (range 1-10+ years). Three years after transplantation 91% of patients had normal white blood counts (WBC), 94% normal haemoglobin (Hb) and 75% normal platelets. Trilineage recovery was complete in 70% (n = 83) at 3 years and 85% (n = 50) at 5 years. Recovery of Hb occurred before WBC and platelet recovery. Approximately 25% of patients displayed an elevated MCV throughout the follow-up period. These long-term results were independent of age, high-dose regimen, number of reinfused stem cells and stem cell source. Double (n = 12) vs single (n = 119) transplantations showed significantly slower trilineage recovery and higher MCV. No secondary graft failure, myelodysplasia or leukaemia was encountered. In conclusion, complete trilineage recovery after HDC followed by ABMT or PSCT occurs slowly. PSCT and ABMT are capable of maintaining long-term haematopoiesis. Slower recovery is seen after double transplantations. The results suggest lasting implications for bone marrow function after autologous transplantation.     

Treatment of severe autoimmune diseases by immunoablative chemotherapy and autologous bone marrow transplantation

The use of bone marrow transplantation for the treatment of refractory autoimmune diseases (AD) is a new concept that is starting to emerge based on many experimental data and some clinical observations obtained in the past 10 years after either allogeneic or, more recently, autologous bone marrow transplantation. Although experimental data demonstrate that allogeneic bone marrow transplantation is effective in treating refractory AD, the treatment-related mortality of such a procedure in humans even in the absence of underlying malignancy, is such that it should only be proposed for patients with refractory AD and an underlying hematological disorder requiring bone marrow transplantation. Autologous bone marrow transplantation, or peripheral hematopoietic stem cell transplantation (HSCT), is currently performed for the treatment of various malignancies including leukemia, lymphomas, or several solid tumors, and this procedure is associated with a low, short-term mortality (<3–5%). Therefore, on the basis of both experimental and clinical data, it has recently been considered an alternative approach to treating autoimmune diseases that are refractory to conventional treatment. Since 1996, an international consensus has emerged and helped to develop some national protocols with clear inclusion criteria, especially in cases of systemic sclerosis, vasculitis, lupus erythematosus, inflammatory myositis, and rheumatoid arthritis that are refractory to conventional treatment. The aim of these phase I–II pilot studies is to define the feasibility of this new procedure, which should still be considered as experimental. Data reporting is an integral part of the protocol. For the first 145 patients reported to the Basel registry, the mortality rate associated with the procedure is 8% (similar to that for non-Hodgkin's lymphoma), well below the estimated death probability at 6 months and 5 years for most of the diseases thus far treated.     

In vivo coagulation activation following infusion of highly purified factor XI concentrate

The use of factor XI concentrates has been associated with thrombosis. Plasma markers of coagulation activation were measured before and 30, 60, 120 and 240 min after six infusions of the BPL factor XI concentrate. Five studies were completed before surgical intervention, one was undertaken in a patient with an intracerebral haemorrhage. Significant elevation of levels of fibrinopeptide A (FpA) ( P  < 0.05) and thrombin–antithrombin (TAT) were demonstrated following six infusions and prothrombin fragment F1.2 following four. Levels of all three markers had risen 60 min following concentrate administration and FpA levels remained elevated throughout the study period. Levels of D-dimer rose in four patients at 240 min. These results indicate significant thrombin generation by 60 min and subsequent plasmin generation consistent with coagulation activation by the factor XI concentrate. The greatest elevation of activation markers was seen in those subjects with pre-existing coagulation activation. We advise caution in the use of these products and awareness of the risks in patients who may already have activated coagulation states.     

The gamma subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon gamma, and transforming growth factor beta 1

The interleukin-2 receptor gamma (IL-2R gamma) chain is a newly recognized component of the IL-2R of lymphoid cells that is required for their response to IL-2. We investigated the expression of IL-2R gamma protein in human monocytes by Western blot analysis using an antiserum specific for IL-2R gamma. We found that IL-2R gamma subunit is constitutively expressed in human monocytes and upregulated by the monocyte-activating factors IL-2 and interferon gamma (IFN gamma). Furthermore, we show that transforming growth factor beta 1 (TGF beta 1) downmodulates, in a dose-dependent manner, basal and IL-2-induced, but not IFN gamma-induced, IL-2R gamma chain expression, and this effect may be responsible for TGF beta 1 suppressive activity on IL-2- activated monocytes. Overall, these results show that the expression of the IL-2R gamma subunit in human monocytes is tightly regulated by the cytokine network, suggesting a critical role played by this protein on monocyte activation.     

Gamma-interferon and tumor necrosis factor production after bone marrow transplantation is augmented by exposure to marrow fibroblasts infected with cytomegalovirus

After bone marrow transplantation (BMT), mortality from viral infections such as cytomegalovirus (CMV) remains high. Gamma-Interferon (gamma IFN) and tumor necrosis factor (TNF) are produced constitutively after BMT and have anti-viral properties. To study the effects of these cytokines on CMV interaction with host cells, we have used patient marrow fibroblasts since marrow stroma is a target for CMV infection correlating with myelosuppression in vivo. Both gamma IFN and TNF are constitutively produced by recipient CD3+ and CD16+ lymphocytes, but not by their marrow fibroblasts. Secretion by peripheral blood mononuclear cells is increased if they are cultured with host fibroblasts infected with CMV in vitro and the levels of gamma IFN and TNF produced are within the range that protects fresh fibroblasts from CMV infection. Constitutive secretion of cytokines by lymphocytes declines by 8 weeks after BMT, a time when the risk of CMV disease increases sharply. The in vitro phenomenon that we have described needs to be evaluated in correlative studies on individual BMT recipients to determine whether such a cytokine-mediated defense mechanism against CMV may operate in vivo.