Synergistic antiproliferative effect of recombinant interferon-gamma with recombinant interferon-alpha on chronic myelogenous leukemia hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM)

Recombinant interferons, alpha (rIFN-alpha) and gamma (rIFN-gamma), have been demonstrated to have significant antitumor activity as single agents in the treatment of chronic myelogenous leukemia (CML). Due to their possible synergistic efficacy, a combination rIFN therapy in CML has been proposed. To establish a biologic basis for this, we have studied the suppressive effects of rIFN-alpha and rIFN-gamma on the in vitro growth of CML-derived progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, CFU-GM), the optimal conditions for rIFN synergism, and the possible role of hematopoietic accessory cells (T-lymphocytes and monocytes- macrophages) in mediating rIFN-induced growth inhibition. When added to unseparated bone marrow cells, rIFN-alpha and rIFN-gamma significantly reduced colony formation, with 50% inhibition occurring at 71 and 186 U/mL for CFU-GEMM, 40 and 152 U/mL for CFU-Mk, 222 and 1,458 U/mL for BFU-E, and 119 and 442 U/mL for CFU-GM, respectively. A small amount of rIFN-gamma (5 U/mL) acted synergistically with increasing doses of rIFN- alpha, and the values of 50% inhibitory concentrations fell outside the lower limit (10 U/mL) used in our experiments. This synergy was evident even when rIFN-gamma was added 72 hours after the initiation of cultures; however, it was completely lost when the target cells were depleted of accessory cells. When a low dose of rIFN-alpha (5 U/mL) was added to rIFN-gamma, the 50% inhibitory concentration values were decreased up to tenfold. These studies (1) confirm that CML-derived hematopoietic progenitors are responsive to the suppressive activity of both rIFN-alpha and rIFN-gamma in vitro, (2) demonstrate that different mechanisms are responsible for the suppressive activity of the two rIFNs, and (3) characterize their synergistic interaction, providing a basis for future clinical trials aimed at investigating combination rIFN therapy in CML.     

Effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM) from patients with myelofibrosis with myeloid metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder due to clonal expansion of a pluripotent hematopoietic progenitor cell with secondary marrow fibrosis. No definitive treatment has as yet been devised for this condition, which shows a marked variability in clinical course. To evaluate whether excessive hematopoietic progenitor cell proliferation could be controlled by recombinant human interferon alpha (rIFN-alpha) and gamma (rIFN-gamma), we studied the effects of these agents on the in vitro growth of pluripotent and lineage-restricted circulating hematopoietic progenitor cells in 18 patients with MMM. A significant increase in the growth (mean +/- 1 SEM) per milliliter of peripheral blood of CFU-GEMM (594 +/- 253), CFU-Mk (1,033 +/- 410), BFU-E (4,799 +/- 2,020) and CFU- GM (5,438 +/- 2,505) was found in patients as compared with normal controls. Both rIFN-alpha and rIFN-gamma (10 to 10(4) U/mL) produced a significant dose-dependent suppression of CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM growth. Concentrations of rIFN-alpha and rIFN-gamma causing 50% inhibition of colony formation were 37 and 163 U/mL for CFU-GEMM, 16 and 69 U/mL for CFU-Mk, 53 and 146 U/mL for BFU-E, and 36 and 187 U/mL for CFU-GM, respectively. A marked synergistic effect was found between rIFN-alpha and rIFN-gamma: combination of the two agents produced inhibitory effects greater than or equivalent to those of 10- to 100- fold higher concentrations of single agents. These studies (a) confirm that circulating hematopoietic progenitors are markedly increased in MMM, (b) indicate that these presumably abnormal progenitors are normally responsive to rIFNs in vitro, and (c) show that IFNs act in a synergistic manner when used in combination. Because rIFN-gamma can downregulate collagen synthesis in vivo, this lymphokine could be particularly useful in the treatment of patients with MMM.     

Effects of recombinant interferons on human megakaryocyte growth

Interferons (IFNs) have been shown to suppress the proliferation of human pluripotent and single-lineage hematopoietic progenitor cells. Treatments with IFNs have reduced platelet counts in patients with myeloproliferative disorders (MPD) but have not altered platelet counts in patients with healthy marrows. We assessed recombinant alpha and gamma IFN (rIFN-alpha and rIFN-gamma) preparations for their effect on the growth of marrow megakaryocytes (MKs) from normal donors and from patients with MPD. In addition, the interactions of recombinant interleukin 3 (rIL-3) recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) with the suppressive effects of rIFNs were examined. The addition of rIFN-alpha to liquid cultures resulted in a dose-dependent inhibition of normal marrow and MPD marrow MK growth. Inhibition with rIFN-gamma was only observed in normal marrows at 2500 U/ml and 12,500 U/ml; and rIFN-gamma unexpectedly stimulated MK growth in some culture conditions. Thirty units per milliliter rIL-3 overcame the inhibitory effect of rIFN-alpha on MK growth, but rGM-CSF or 3 U/ml rIL-3 did not. These studies with rIFN-alpha demonstrate that MPD marrow MKs and their precursor cells are no more sensitive to rIFN-alpha than are normal marrow MKs. Clinically, rIFN-gamma would be expected to be less effective than rIFN-alpha at controlling thrombocytosis in patients with MPD.     

Diversity in inhibitory effects of IFN-gamma and IFN-alpha A on the induced DNA synthesis of a hairy cell leukemia B lymphocyte clone reflects the nature of the activating ligand

A hairy cell leukemia population was used as a clonal model for studying the direct immunomodulatory effects of recombinant interferon- alpha A (rIFN-alpha A) and rIFN-gamma on human B-cell proliferation. The leukemic cell population KON was notably quiescent when incubated in medium alone but was induced to significant in vitro DNA synthesis when cultured with any of four activators of human B cells: anti-IgM antibody, Staphylococcus aureus cells (SAC), phorbol myristate acetate (PMA), or B-cell growth factor (BCGF). While both rIFN-gamma and rIFN- alpha A exhibited suppressive effects on these responses, their inhibitory patterns were distinct and reciprocal. Thus, rIFN-gamma exclusively suppressed anti-IgM-and SAC-induced leukemic DNA synthesis, and rIFN-alpha A significantly suppressed only PMA- and BCGF-induced DNA synthesis. The effects of the rIFN preparations were ablated in the presence of IFN type-specific monoclonal antibodies. Kinetic analyses and pulsing studies revealed that inhibition was most notable when cells were exposed concomitantly to IFN and the activating ligand. That the diverse effects of IFN-gamma and IFN-alpha A are manifested on a single B-cell clone was confirmed by Southern blot analysis of restriction enzyme-digested KON cell DNA with a JH-specific probe. These studies suggest that the therapeutic potential of the two types of IFN may be influenced by the nature of the extracellular ligands in the leukemic mileau that promote leukemic clonal expansion.     

Megakaryocytic differentiation capacity of human pluripotent bone marrow progenitor cells CFU-GEMM in vitro after cryopreservation

The effect of cryopreservation on the pluripotent haemopoietic progenitors CFU-GEMM as well as on the megakaryocytic (CFU-Mk), erythroid (BFU-E) and granulocytic-monocytic (CFU-GM) progenitor cells was analyzed. Progenitor cell recovery after freezing, as determined in 5 experiments, averaged 89% for CFU-GEMM (range: 63% - 194%), 85% for CFU-Mk (range: 62% - 96%), 92% for BFU-E (range: 43% - 174%) and 60% for CFU-GM (range: 31% - 93%). Immunological analysis of individual mixed colonies using a double labelling immunoalkaline phosphatase slide technique and monoclonal antibodies against megakaryocytic and granulocytic cells revealed megakaryocytic cells in more than 79% (range: 73% - 94%) and 84% (range: 75% - 87%) of mixed colonies before and after freezing, respectively. Our results indicate that cryopreservation of human bone marrow cells does not alter the megakaryocytic differentiation capacity of the haemopoietic progenitor cells CFU-GEMM and CFU-Mk in vitro.     

Combinations of recombinant human interferons and retinoic acid synergistically induce differentiation of the human promyelocytic leukemia cell line HL-60

The human acute promyelocytic leukemia cell line HL-60 is induced to differentiate into morphologically and functionally mature monocytelike cells by incubation with a combination of 10 nmol/L retinoic acid (RA) and various concentrations of recombinant immune interferon (rIFN- gamma). These induced cells show marked increases in antibody-dependent cellular cytotoxicity (ADCC), antibody-coated erythrocyte (EA) rosettes, nonspecific esterase, and 5'-nucleotidase activity. rIFN- gamma alone at concentrations of 10 to 1,000 U/mL has essentially no effect on morphological maturation, nitroblue tetrazolium reduction, and immunophagocytosis. However, rIFN-gamma at these concentrations increases EA rosetting in a concentration-dependent manner that is not affected by 10 nmol/L RA. At a concentration of 1,000 U/ml, rIFN-gamma induces moderate increases in nonspecific esterase, 5'-nucleotidase, and ADCC. These parameters are markedly increased by the addition of 10 nM RA, a concentration which alone has no effect on these markers. Based on units of antiviral activity, rIFN-gamma is tenfold more active than rIFN-alpha D in inducing EA rosettes and 40-fold more active in inducing nitroblue tetrazolium reduction and immunophagocytosis. These results, indicating that combinations of rIFN-gamma or rIFN-alpha and RA synergistically induce differentiation of HL-60, suggest that this combination may have clinical utility in the treatment of patients with certain leukemias.     

Megakaryocyte differentiation capacity of human pluripotent bone marrow progenitor cells CFU-GEMM in vitro after cryopreservation*

The effect of cryopreservation on the pluripotent haemopoietic progenitors CFU-GEMM as well as on the megakaryocyte (CFU-Mk), erythroid (BFU-E) and granulocytic-monocytic (CFU-GM) progenitor cells was analyzed. Progenitor cell recovery after freezing, as determined in 5 experiments, averaged 89% for CFU-GEMM (range: 63% - 194%), 85% for CFU-Mk (range: 62% - 96%), 92% for BFU-E (range: 43% - 174%) and 60% for CFU-GM (range: 31% - 93%). Immunological analysis of individual mixed colonies using a double labelling immunoalkaline phosphatase slide technique and monoclonal antibodies against megakaryocytic and granulocytic cells revealed megakaryocyte cells in more than 79% (range: 73% - 94%) and 84% (range: 75% - 87%) of mixed colonies before and after freezing, respectively. Our results indicate that cryopreservation of human bone marrow cells does not alter the megakaryocytic differentiation capacity of the haemopoietic progenitor cells CFU-GEMM and CFU-Mk in vitro.     

In vitro and in vivo effects of recombinant interferon gamma on the growth of hematopoietic progenitor cells from patients with myelodysplastic syndrome

Recombinant interferon gamma (rIFN-gamma) has been shown to have antiproliferative effects on normal and leukemic hematopoietic cells, to induce cell differentiation and to modulate hematopoietic growth factor production. We have studied the effects of rIFN-gamma on the growth of hematopoietic progenitors from 3 patients with myelodysplastic syndrome who were treated with rIFN-gamma (0.01 mg/m2 given subcutaneously three times a week) as part of an Italian pilot study. When bone marrow cells were cultured in semisolid medium in the continuous presence of rIFN-gamma (10-10(4) U/ml), inhibition of colony formation was the most common response. However, an enhancement of hematopoietic progenitor growth was observed in one patient at the lowest concentration tested (10 U/ml). Preincubation of bone marrow mononuclear cells with low concentrations of rIFN-gamma in suspension culture for 5 days induced or enhanced in vitro colony formation in two cases; again, higher concentrations resulted in inhibition of hematopoietic progenitor growth. Two patients showed a slight improvement of in vitro progenitor growth after one month of treatment with rIFN-gamma. Although preliminary, these data indicate that rIFN-gamma may have both stimulatory and inhibitory effects on myelodysplastic hematopoiesis, depending on both the effective concentrations and the interactions with accessory cells.     

Influence of human recombinant interferon-alpha and interferon-gamma on bone marrow progenitor cells of HIV-positive individuals.

As a result of a pathophysiologically unexplainable bone marrow failure, most patients with progressive stages of human immunodeficiency virus (HIV) infection develop anemia, leukopenia, and thrombocytopenia. Besides the possibility of immune-mediated cytolysis or of direct viral infection of hemopoietic progenitor cells, the inhibitory influence of cytokines, for example interferon-alpha (IFN-alpha) and IFN-gamma, on hemopoiesis of HIV-infected patients might be considered as one parameter that contributes to myelosuppression. Therefore, progenitor cells from the bone marrow of HIV+ and HIV- persons were exposed to increasing concentrations of recombinant human IFN-alpha and IFN-gamma in methylcellulose assays. The colony formation of pluripotent (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells was inhibited by both interferons. The 50% inhibitory doses (ID50) of IFN-alpha were 125.6 U/mL and 131.5 U/mL for BFU-E from HIV-infected persons and normal controls, respectively; the corresponding ID50 of IFN-alpha for CFU-GM growth was 1095.8 U/ml and above 3000 U/ml. When IFN-gamma was studied the ID50 was 341.7 and 2794.6 U/ml for BFU-E from HIV-infected and healthy individuals, respectively, while the ID50 for CFU-GM was above the highest dose levels in both groups (greater than 3000 U/ml). The ID50 for CFU-GEMM was below the lowest dose levels of IFN alpha and IFN gamma tested in both groups (less than 10 U/ml). The inhibitory effects could be specifically neutralized by monoclonal antibodies against IFN-alpha and IFN-gamma, thus confirming that the suppressive effects were due to the cytokines used.     

In vitro growth of bone marrow-derived multipotent and lineage-restricted hematopoietic progenitor cells in myelodysplastic syndromes

The aim of the present study was to evaluate the incidence of bone marrow-derived multipotent (CFU-GEMM), megakaryocytic (CFU-Mk), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in 22 patients with myelodysplastic syndromes (MDS), and to investigate the role of hematopoietic accessory cells (T-lymphocytes and monocytes) as a possible cause of growth derangement. As compared to normal controls (n = 15), growth values in the 22 patients (mean +/- SEM) were significantly reduced for CFU-GEMM (0.4 +/- 0.1 versus 7 +/- 1, P less than 0.0005), CFU-Mk (1.4 +/- 0.5 versus 18 +/- 4, P less than 0.0005), BFU-E (2.2 +/- versus 40 +/- 6, P less than 0.0005), and CFU-GM (19 +/- 5 versus 65 +/- 10, P less than 0.0005). The growth of CFU-GEMM was abnormal at an early stage in the clinical development of MDS, sometimes even when CFU-GM formation was still normal. Colony-formation was unaffected by removal of hematopoietic accessory cells. Although no correlation was found between the incidence of lineage-restricted progenitors and the degree of peripheral cytopenia, derangement of colony growth was more pronounced in patients with worse prognosis. We conclude that: (i) the grossly defective CFU-GEMM growth supports the concept of MDS as clonal disorders of hematopoietic multipotent stem cells; (ii) a progressive impairment of in vitro hematopoiesis occurs in association with the clinical progression of the myelodysplastic syndromes.     

Therapy of chronic myelogenous leukemia with recombinant interferon- gamma

Recently, we reported that recombinant interferon-alpha (rIFN-alpha) can induce hematologic remissions and cytogenetic improvement in newly diagnosed Philadelphia (Ph)-positive chronic myelogenous leukemia (CML) patients. Although IFN-gamma is a structurally distinct molecule, this agent suppresses in vitro hematopoietic progenitor cells in a fashion similar to that of IFN-alpha. Therefore, we initiated a study of rIFN- gamma at doses of 0.25 to 0.5 mg/m2/d intramuscularly in patients with Ph-positive benign-phase CML. Twenty-six of 30 patients entered in the study were evaluable. Six patients have achieved a complete hematologic response; four, a partial hematologic response. The median follow-up period of patients who are in complete remission is 7.5 months (range, 5 to 12 months). No relapses have occurred among the complete responders. So far, five patients have had cytogenetic improvement with emergence of 5% to 45% diploid cells in the bone marrow. Fever and flulike symptoms were the most common side effects, with partial tolerance often developing after about 1 week. The majority of patients tolerated therapy with minimal change in performance status. In conclusion, rIFN-gamma has demonstrated clinical activity in CML. On the basis of these observations and the in vitro synergistic growth- inhibitory effects of IFN-alpha and IFN-gamma, we have started trials of combination IFN therapy in CML patients.     

Lymphokine(s) from isolated T lymphocyte subpopulations support multilineage hematopoietic colony and megakaryocytic colony formation

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.     

Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential- colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC- IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40- fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.     

Surface antigenic determinants on human pluripotent and unipotent hematopoietic progenitor cells

RFB-1 is a monoclonal antibody previously shown to react with granulocyte-monocyte progenitors (CFU-GM) and immature lymphoid cells in human bone marrow. RFB-HLA-DR is a monoclonal antibody that reacts with HLA-DR (la-like) antigens. The present study shows that the bone marrow subset reactive with both RFB-1 and RFB-HLA-DR contains all the cells that give rise to mixed hematopoietic colonies (derived from CFU- GEMM; a pluripotent human progenitor cell) as well as to megakaryocytic (megakaryocyte-CFU-derived) and erythropoietic (derived from erythroid burst-forming units, BFU-E) colonies, as shown by fluorescence- activated cell sorting and complement-mediated cytotoxicity. These results indicate that CFU-GEMM, BFU-E, and megakaryocyte-CFU express RFB-1 and la-like antigens. RFB-1 antigen is also expressed on erythroid colony-forming units (CFU-E). RFB-1 and RFB-HLA-DR are useful reagents in the study of hematopoietic stem cells.     

Recombinant alpha-interferon inhibits colony formation of bone marrow fibroblast progenitor cells (CFU-F).

Alpha-Interferon (IFN-alpha) has been shown to inhibit colony formation of hematopoietic progenitor cells, including colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), day 7 colony-forming unit granulocyte-macrophage (CFU-GM), day 14 CFU-GM, burst-forming unit erythroid (BFU-E), pluripotent stem cells (CFU-S), and colony-forming unit megakaryocyte (CFU-MK). The present study was designed to see whether IFN-alpha also has inhibitory effects on bone marrow fibroblast progenitors (CFU-F). We found that IFN-alpha exerted a significant inhibitory effect on both rabbit and human CFU-F formation. Inhibition of human marrow CFU-F formation by alpha interferon was unaffected by removal of 98% of monocytes/macrophages and T lymphocytes from light density marrow cells. This finding suggests that IFN-alpha probably exerts a direct inhibitory effects.     

Megakaryocytic cells in mixed haemopoietic colonies (CFU-GEMM) from the peripheral blood of normal individuals

The cellular composition of mixed haemopoietic colonies CFU-GEMM from the peripheral blood of normal individuals was investigated using both an improved in vitro culture assay and a monoclonal antibody reactive with the earliest megakaryocytic precursor cells. The frequency of CFU-GEMM was found to be 22 +/- 7/ml blood (mean +/- SE; n = 8) and 4.9 +/- 1.8/10(6) mononuclear cells. Double-labelling of single colonies with monoclonal antibodies against megakaryocytic as well as granulocytic cells revealed that only 18% of mixed colonies from the peripheral blood contained megakaryocytic cells as compared to 87% of mixed colonies from the bone marrow, while no difference existed with regard to granulocytic cells. It is concluded that circulating multilineage progenitor cells either require different culture conditions to express their megakaryocytic component or, alternatively, that the circulating pluripotent stem cell pool mainly consists of bipotent rather than multipotent progenitor cells.     

Development of pluripotent hematopoietic progenitor cells in the human fetus

Pluripotent hematopoietic progenitor cells (CFU-GEMM), myeloid progenitor cells (CFU-GM), and erythroid progenitors (BFU-E) were studied in midtrimester human fetuses using the mixed colony assay. All three progenitor cell populations were detected at high levels in the fetal liver from 12 to 23 wk of gestation. Stem cells were first observed in the bone marrow at 15-16 wk of gestation, although bone marrow cultures from earlier fetuses showed heavy growths of stromal cells. Spleen cultures first showed growth of stem cells at 18-19 wk, but fetal thymus showed no hematopoietic activity. Peripheral blood from four fetuses aged 13, 18, 20, and 21 wk showed very high levels of all 3 progenitor cells. The results demonstrate that hematopoietic development in the human fetus parallels that of the mouse. The observation that stromal cell development in the bone marrow precedes the appearance of hematopoietic progenitor cells suggests that they may be closely involved in stem cell growth.     

Lymphokine-activated killer cells selectively kill tumor cells in bone marrow without compromising bone marrow stem cell function in vitro

Although it has been demonstrated that lymphokine-activated killer (LAK) cells kill tumor cells in a selective way without being toxic to a variety of normal cells, contradictory results exist about the possible toxicity of natural killer (NK) and LAK cells for hematopoietic progenitor cells. Therefore, the cytolytic activity and growth inhibitory effects of LAK cells on normal bone marrow progenitor cells and the ability of LAK cells to eliminate neoplastic hematopoietic cells from populations of bone marrow cells in vitro was studied. The results of these experiments show the following: (1) LAK cells have little cytolytic activity against normal bone marrow cells; (2) normal bone marrow cells fail to cold target compete for the killing of the hematopoietic tumor cell lines K562 and HL60 or freshly frozen acute myelocytic leukemia (AML) blast cells by LAK cells; (3) LAK cells inhibit the growth of K562 and HL60 to more than 90% in clonogenic assays; (4) LAK cells have no inhibitory effect on hematopoietic progenitor growth in CFU-GM (colony-forming unit- granulocytes, macrophages), CFU-E (colony-forming unit-erythrocytes), BFU-E (burst-forming units-erythrocytes), or CFU-GEMM (colony-forming unit-granulocytes, erythrocytes, macrophages, megakaryocytes) assays. These results indicate that LAK cells have low toxicity for normal bone marrow and that LAK activity against tumor cells is not adversely affected by the presence of normal bone marrow cells. The differences in cytolysis and growth inhibition of neoplastic hematopoietic cells and hematopoietic progenitor cells by LAK cells in vitro could create a therapeutic index that might allow the use of LAK cells for cleansing of the autologous bone marrow graft and for adjuvant therapy in combination with autologous bone marrow transplantation without compromising the reconstitution of the bone marrow in the host.     

Morphology of cells grown in the CFU-GEMM tissue culture assay from mononuclear cells obtained from peripheral blood and bone marrow of normal volunteers

In ten healthy volunteers studies were done to assess the morphology, immunocytology, and cytochemical properties of mononuclear hematopoietic stem cells isolated from normal human peripheral blood and bone marrow grown in semisolid matrix mixed lineage colony-forming unit (CFU-GEMM) culture. In three volunteers, peripheral blood and bone marrow samples were collected simultaneously; the progeny were remarkably similar in each of the three paired cultures, although macrophages were more numerous in the bone marrow cultures. Three peripheral blood samples were cultured following frozen storage with 10% DMSO at-150 degrees C for approximately 5 months. Megakaryopoiesis was present in each case, demonstrating the full hematopoietic potential of previously frozen peripheral blood mononuclear cells. Four other peripheral blood samples and one bone marrow sample were cultured and, in each case, cells of all hematopoietic cell lines were present. Bone marrow, peripheral blood mononuclear cells, and previously frozen peripheral blood mononuclear cells grown in the CFU-GEMM tissue culture assay showed the presence of granulocyte-macrophage, erythroid, and megakaryocytic cell lines.     

Influence of tumor necrosis factor-alpha on the modulation by interferon-gamma of HLA class II molecules in human thyroid cells and its effect on interferon-gamma binding

Cytokines are important modulators of immunological reactions, but it has been postulated that they might act on other unrelated epithelial cells. We studied the effects of recombinant interferon-gamma (rIFN gamma) and recombinant tumor necrosis factor-alpha (rTNF alpha) on normal human thyroid cells. We found that the combination of these two cytokines enhanced HLA class II molecule expression on these cells compared with the effect of rIFN gamma alone. This was proven by both immunofluorescence as well as a more sensitive and quantitative RIA. rTNF alpha alone had no effect on HLA class II molecule induction on the same thyrocytes, suggesting a synergistic rather than an additive action in combination with rIFN gamma. The addition of 600 U/ml rTNF alpha to low dose rIFN gamma (10 U/mL) enhanced class II expression by 50%, as quantified by RIA. We also demonstrated that normal thyrocytes possess distinct receptors for the two cytokines and that rTNF alpha probably augments IFN gamma binding, since it increased when the cells were first incubated with rTNF alpha. This increased binding provides an explanation for the synergistic action of rTNF alpha in enhancing class II molecule expression by rIFN gamma. We conclude that the presence of receptors for these cytokines on human thyroid cells gives a direct demonstration of their potential biological action on cells normally not involved in the immunological circuit. The phenomenon might also explain their direct or indirect involvement in vivo, such as in influencing inappropriate HLA class II molecule expression in epithelial cells affected by autoimmunity.