Enhancement of human T lymphocyte colony formation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocyte lymphocyte colony formation in vitro were investigated. The number of T lymphocyte colonies was increased 4-5 times over that of controls by the addition of TPA (10(-7) - 10(-9) M) to phytohemagglutinin (PHA)-containing cultures. Few colonies were observed when stimulated with TPA in the absence of PHA. In the cultures containing a sufficient amount of exogenous T cell growth factor (TCGF), the enhancement of T lymphocyte colony formation by TPA was not observed. TPA enhanced TCGF production by peripheral lymphocytes stimulated with PHA. The optimal concentrations of TPA for T lymphocyte colony formation were similar to those for TCGF production. These findings suggest that TPA enhanced T lymphocyte colony formation by stimulating endogenous TCGF production. Interestingly, T lymphocyte colony formation was not inhibited even at high concentrations of TPA that usually inhibit myeloid and erythroid colony formation. This difference may be due to different sensitivities to TPA between T lymphocyte colony-forming cells and myeloid and erythroid colony-forming cells.     

Inhibition of Normal Human Granulopoiesis in Vitro by Non-B Non-T Lymphocytes

S ummary Normal non-adherent mononuclear cells were shown to inhibit colony formation by normal human marrow cells cultured for 7 d in semi-solid agar. Inhibition was the same using cells from the marrow donor or from an unrelated normal subject, and was shown to be dose-dependent over the range of 4 × 10⁵ to 6 × 10³ mononuclear cells per 1 × 10⁵ marrow cells plated. Inhibition was not seen in 14 d cultures, and it is postulated that colony-forming cells sensitive to lymphocyte inhibition belonged to the population known to give rise to colonies after 7 d in culture. Cell fractionation studies showed that inhibition was due to non-B non-T lymphocytes, purified B cells or T cells being neither inhibitory nor stimulatory. Inhibition was only shown with intact viable lymphocytes and it was not possible to extract inhibitory activity from the cells, or to produce inhibition by media conditioned by lymphocytes. The effect was apparently due to a direct action on colony-forming cells in the marrow and was not due to inhibition of colony stimulating activity (CSA) production, or to absorption or inactivation of CSA. These results emphasize the need to include appropriate controls when looking for possible cell-mediated inhibitors in disease states, particularly when 7 d cultures are used.     

A method for cloning T-lymphocytic precursors in agar.

A new technique for the culture of T-lymphocytic colonies is reported. The method may be regarded as a human lymphocyte precursor cell assay, as is the myeloid colony culture for granulocyte-macrophage progenitors. The colonies arise under the simultaneous stimulation of phytohemagglutin and a leukocyte feeder. A linear relationship is found between colony numbers and cell numbers plated. The colonies represent aggregates of lymphoblast-like cells, the majority of which are capable of E-rosette formation, are responsive in mixed lymphocyte cultures, and do not exhibit surface immunoglobulins. Their density distribution profile is very similar to that of myeloid colony-forming cells. The finding that most of these colony-forming cells are recovered in the so-called lymphocyte-free stem cell fraction following density fractionation suggests that they originate from a lymphocytic precursor.     

Effect of platelet-derived growth factor and bone marrow-conditioned medium on the proliferation of human bone marrow-derived fibroblastoid colony-forming cells

The effects of various human sera, platelet lysates and platelet-derived growth factor (PDGF) on the proliferation of human bone marrow-derived fibroblastoid colony-forming cells (CFU-F) were examined. We obtained nearly identical growth curves of fibroblastoid colonies with sera, platelet lysates and PDGF as stimulants and concluded that PDGF was a main growth factor for CFU-F in human serum. In contrast to colony size, CFU-F number was irrelevant to the concentration of PDGF. Removal of culture medium containing hemopoietic cells after short-term incubation of bone marrow cells reduced both colony number and size in CFU-F cultures. When each of bone marrow-conditioned medium (BMCM), peripheral blood mononuclear cells (MNC) and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) was added to the cultures, CFU-F number and colony size recovered. The role of PDGF and the factors present in BMCM, MNC and PHA-LCM in the growth of CFU-F and their precursor cells were discussed.     

Humoral factors modulating growth of granulocyte macorphage progenitor cells.

The kinetic of production of colony-stimulating activity (CSA) inducing mouse and human colony-forming cells (CFU-C) was tested in different human leukocyte culture systems. Stimulated and unstimulated cultures of spleen single cell suspensions, peripheral mononuclear leukocytes and acute monocytic leukemia (AMoL) cells were investigated. With the exception of the AMoL cells, stimulated cultures always revealed higher CSA levels than unstimulated controls. The spleen cell cultures exhibited the highest overall activity showing three molecular species of 70,000, 35,000 and 10,000 daltons activating human CFU-C to form colonies in the agar culture system. Furthermore it could be demonstrated that colony formation could be inhibited by low molecular weight fibrinogen degradation products obtained by digestion of fibrinogen with granulocyte-derived elastase.     

A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B- cell colony formation and an inhibitory effect of IL-1 alpha

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7- induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.     

Characterization of normal peripheral blood lymphocyte colony-forming cells: cell cycle status, surface markers, and cellular growth requirements

We performed a series of studies to further clarify the nature of lymphocyte colony-forming cells (CFC) from normal peripheral blood. Mononuclear cells were separated into E-rosette-enriched (E+) and E- rosette-depleted (E-) populations and cultured in methylcellulose with conditioned media and irradiated mononuclear cells. Linear plating relationships were obtained with plating efficiencies of 0.26% +/- .02% (mean +/- SE) for E+ CFC and 0.18% +/- .02% for E- CFC. Cells in E+ colonies were T lymphocytes and in E- colonies were B lymphocytes as determined by cell surface marker analysis. Using the thymidine suicide technique, approximately one-half of CFC were found to be in cycle at any moment, and plating efficiencies and cell cycle status of E+ CFC were not changed by preincubation with PHA in liquid culture for 48 hr. Using antibody complement-mediated cytotoxicity, E+ CFC were found to be T101+, OKT3+, and Ia-, while E- CFC were OKT3- and Ia+. Using monocyte-depleted populations obtained by sedimentation at unit gravity, lymphocyte colony growth was absent in monocyte-depleted fractions, and optimal growth occurred with 40% monocytes in culture. In contrast to some previous studies, we find that lymphocyte CFC originate from a small, cycling population of cells bearing mature T or B lymphocyte markers. Entry into cell division, however, does not confer colony-forming capacity on lymphocytes. Monocytes are critical to growth of E+ CFC, and cultures severely depleted of monocytes would not be expected to form colonies.     

In vitro and in vivo effects of MDP-Lys(L18) on mouse megakaryocyte progenitor cells (CFU-Meg).

We investigated the in vitro and in vivo effects of MDP-Lys(L18), a derivative of muramyl dipeptide (MDP), on megakaryocyte progenitor cells (megakaryocyte colony-forming units, CFU-Meg) in the mouse bone marrow and spleen. When CFU-Meg culture was performed with a suboptimum concentration (2%) of pokeweed mitogen-stimulated mouse spleen-conditioned medium (PWM-SCM), addition of 0.1-20 micrograms/ml of MDP-Lys(L18) increased the number of megakaryocyte colonies. The size of the megakaryocyte colonies (the number of megakaryocytes per colony) was also significantly increased by the addition of MDP-Lys(L18) under the same culture conditions in comparison with cultures without MDP-Lys(L18). MDP-Lys(L18) itself did not stimulate megakaryocyte colony formation without PWM-SCM, and it failed to enhance megakaryocyte colony formation in cultures with an optimum PWM-SCM concentration (10%). Furthermore, no effect of MDP-Lys(L18) was observed in cultures of phagocytic cell-depleted bone marrow cells. However, MDP-Lys(L18) enhanced megakaryocyte colony formation in cultures of T-lymphocyte-depleted bone marrow cells. The culture supernatant from a macrophage cell line, J774.1, plus MDP-Lys(L18) enhanced in vitro megakaryocyte colony formation in cultures with a suboptimum PWM-SCM concentration. Although interleukin 1 (IL-1)beta in the culture supernatant of J774.1 plus MDP-Lys(L18) was increased in a dose-dependent manner in response to MDP-Lys(L18), the effect of the culture supernatant was not blocked by an anti-IL-1 antibody, and IL-1 beta failed to enhance megakaryocyte colony formation in the presence of suboptimum PWM-SCM levels. The enhancement of megakaryocyte colony formation by MDP-Lys(L18) could be neutralized, however, by an anti-interleukin 6 (IL-6) antibody. Intraperitoneal administration of MDP-Lys(L18) (100 micrograms daily for 3 days) significantly increased the number of bone marrow and spleen megakaryocyte colonies at 24 to 72 h after the final injection. These in vitro and in vivo observations strongly suggest that MDP-Lys(L18) indirectly enhances the proliferation and differentiation of mouse CFU-Meg via colony-stimulating factor(s) other than IL-1, probably as a result of the stimulation of macrophages to produce IL-6.     

Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor.

Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.     

Growth hormone stimulation of normal and leukemic human T-lymphocyte proliferation in vitro

In order to investigate the effect of human growth hormone on T lymphocytes, we utilized a clonogenic assay for mitogen-responsive human peripheral blood T lymphocytes. Lymphocytes were purified by density gradient centrifugation and incubated in the presence of phytohemagglutinin using a two-layer agar technique for CFU- T- lymphocyte culture. Nanogram concentrations of human growth hormone, ovine prolactin, human chorionic somatomammotropin, or growth hormone fragment were added to cell cultures. Growth hormone potentiated normal T-cell colony formation in a species-specific manner. Cells from a homogeneous T-lymphoblast line derived from a patient with a T-cell variant of hairy cell leukemia also showed augmentation of colony growth in the presence of human growth hormone. These studies provide evidence for a direct effect of growth hormone on normal and some neoplastic human T cells.     

Marrow culture in diffusion chambers in rabbits: III. Effect of endotoxin and leukocyte products on cell production

Soluble leukocyte products harvested from incubated peritoneal exudate leukocytes, injected intravenously or intramuscularly, increased production of granulocytes and macrophages in marrow in diffusion chambers implanted into the peritoneal cavity of rabbits. Red cell production in the chambers was not consistently affected. Endotoxin increased production of all cell types. Endotoxin tolerance induced by daily injection of endotoxin to host rabbits abolished granulopoietic stimulation by endotoxin given during the culture period but did not diminish the granulopoietic stimulation produced by injected leukocyte products. Attempts to induce tolerance to leukocyte products by daily injections did not reduce the granulopoietic stimulation produced by either endotoxin or leukocyte products injected during the culture period. Intraperitoneally administered leukocytes products markedly inhibited production of all cell types. Endotoxin or leukocyte products given to normal rabbits increased plasma colony stimulating activity (CSA); the increase occurred sooner after leukocyte products than after endotoxin. Endotoxin-tolerant animals showed no rise in CSA after endotoxin, but their response to leukocyte products was normal. Leukocyte products added to agar cultures neither supported nor inhibited colony growth but augmented CSA-stimulated colony production. Endotoxin, leukocyte products, and CSA are different and may interact in regulating granulopoiesis.     

The Effects of Whole Body Irradiation on Serum Colony Stimulating Factor and in Vitro Colony-Forming Cells in the Bone Marrow

S ummary . Using in vitro cultures of mouse bone marrow cells, levels of colony stimulating factor were assayed in serum from mice following whole body irradiation. No significant differences from control levels were noted in the period 1–32 days following 50, 150, 250 or 450 rads. A dose of 250 rads caused a sharp fall in the level of colony-forming cells in the bone marrow followed by regeneration between 6 and 16 days following irradiation.
Six to 8 hours following whole body irradiation, blood polymorphs and serum colony stimulating factor levels were consistently elevated. Splenectomy did not affect this response.
The results suggest that serum colony stimulating factor is unlikely to be the major regulator determining regeneration of in vitro colony-forming cells in the bone marrow following irradiation.     

Enhancement of colony-stimulating activity production by lithium.

Since lithium causes granulocytosis in some patients, its effect upon granulocyte production was investigated using mouse marrow in the agar culture system. When lithium was added to semisolid cultures of mouse marrow, there was no stimulation of colony formation in the absence of colony-stimulating activity (CSA). In addition, lithium did not potentiate the action of already formed CSA. However, lithium did stimulate the production of CSA by lung tissue. Lithium enhancement of CSA production was blocked by puromycin, indicating that lithium action required active new protein synthesis. It was concluded that lithium promoted enhanced granulocyte production in vitro by stimulating the synthesis of CSA.     

Effects of normal mouse serum on the IL-3-induced proliferation of bone marrow cells

Normal mouse serum (NMS) devoid of colony-stimulating factor (CSF) was found to enhance the interleukin 3 (IL-3)-driven colony formation of bone marrow in vitro. Inclusion of NMS in bone marrow colony-forming assays resulted in greatly increased numbers of colonies and clusters following seven days incubation; however, incubation of bone marrow with NMS before the colony-forming assay had no effect on resultant colony number. The levels of serum-enhancing activity (SEA) did not appear to vary significantly with age and in part was species restricted, in that human and guinea pig serum did not enhance mouse bone marrow colony formation. Conversely, NMS had no effect on human bone marrow colony formation. Levels of SEA were found to vary between strains, as did the degree to which bone marrow from various strains was enhanced by the serum. Serum fractionation studies indicated three active fractions with molecular weights of 800-900 Kd, 60-70 Kd, and 20- 30 Kd. The fraction at 800-900 Kd inhibited colony formation at high concentrations and enhanced colony formation on dilution, whereas the two other active fractions contained enhancing activity at all concentrations tested. These results would indicate that normal serum can play a greater role in colony-forming assays than nutritional supplements. The relationship of the SEA factors to other factors that have been reported to modulate bone marrow colony formation is discussed.     

Interleukin 2 stimulates chronic lymphocytic leukemia colony formation in vitro

The requirements of clonogenic cells of B cell-type chronic lymphocytic leukemia (B CLL) for interleukin 2 (IL 2) were analyzed. Using the cells of five patients, we measured IL 2 receptor expression on the cell surface and the colony-forming abilities of the cells in response to IL 2. In four of the cases, significant percentages of the CLL cells expressed IL 2 membrane receptors (as assessed with the monoclonal antibody anti-Tac), indicative of their potential sensitivity to IL 2. Pure recombinant interleukin 2 (r-IL2) was added to colony cultures that also contained the lectin phytohemagglutinin (PHA) or the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to activate the CLL cells. Colony formation completely depended on the presence of r-IL 2 and PHA or TPA in culture, with the exception of one case, in which the addition of IL 2 was not required for colony growth in TPA-supplemented cultures. Twenty-five to fifty units of r-IL 2 per milliliter of culture medium provided optimal stimulation. Under these conditions, a linear relationship was observed between plated cell numbers and colony numbers formed. Morphological and immunologic analysis of colony cells indicated that these were monoclonal CLL cells that had matured toward plasmacellular lymphocytes and plasma cells.     

Impairment of T lymphocyte colony formation in Hodgkin's disease: effect of soluble inhibitory factors on normal T lymphocyte colony formation potential

PHA-induced T lymphocyte colonies from peripheral blood mononuclear cells of untreated Hodgkin's disease (HD) patients and normal healthy donors were assayed by one-step stimulation in microagar capillary cultures. A significant depression in the T cell colony formation was observed in HD patients in comparison with normal healthy donors. Clinical staging of the disease had no influence on this abnormality. Preincubation of HD lymphocytes for 24 h in tissue culture medium did not produce any appreciable recovery in the colony formation potential. However, in the presence of 24-hour culture supernatants of HD mononuclear cells, there was significant inhibition in the colony formation by lymphocytes obtained from normal healthy donors. Significance of these observations is discussed.     

Antitumor activity of interferon against murine osteogenic sarcoma in vitro and in vivo.

Murine interferon inhibited the growth of a continuous line of osteogenic sarcoma cells in tissue culture. Inhibition of tumor cell growth was documented by decreased clone formation in liquid medium, decreased tumor cell counts in monolayer cultures, suppression of colony formation in semi-solid agar, and decreased uptake of 3H-thymidine by the osteogenic sarcoma cells in culture. The capacity of anti-interferon antibody to block the tumor cell growth inhibitory activity of the interferon preparation suggested that interferon itself is the biologically active component of the interferon preparations. In vivo, a 7-day course of 30,000-60,000 units/day of type I interferon prepared in cell cultures either completely inhibited or delayed the appearance of tumors in experimental animals inoculated with osteogenic sarcoma cells by the sc route. The therapeutic efficacy of a preparation of murine sera containing type II interferon as well as other lymphokine activity was compared with the type I interferon preparation. Animals treated with 600 units of type II interferon were protected against tumor development as effectively as with 60,000 units/day of type I.     

Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia

Inhibitory activity in extract from human blood granulocytes was tested on granulocyte-macrophage colony formation in vitro. The inhibition depended on the type of serum used. With mouse BMC and FCS in the cultures, extract corresponding to 2.5 X 10(4) granulocytes/ml reduced the colony number by 35%, and extract from 2 X 10(5) cells caused maximal inhibition (80-90%). With HS and mouse BMC the colony number was reduced by only 11-12%, but stronger inhibition (55%) was observed when the serum concentration was reduced. With both types of sera the total cell number per culture plate was reduced relatively more than the colony number. Human GM-CFC were as sensitive as mouse GM-CFC, and extract from CML granulocytes inhibited less (p less than 0.01) than extract from normal cells. Biochemical studies indicated that the inhibitor is a protein with a molecular weight of 30-60,000. Lactoferrin, a putative inhibitor of CSF production, did not inhibit spontaneous or CSF-induced colony formation in these studies.     

Recombinant interleukin 6 stimulates immature murine megakaryocytes

Human recombinant interleukin 6 (IL-6) was found to stimulate the growth of immature mouse megakaryocytes maximally at 2 ng/ml, leading to significant increases in the number of large megakaryocytes readily detectable by light microscopy. IL-6 did not stimulate megakaryocyte progenitor cells to form colonies of megakaryocytes, but potentiated megakaryocyte colony formation when added in the presence of interleukin 3. The stimulation could be neutralized by an anti-IL-6 serum. The data indicate that IL-6 is a potent differentiation stimulus of megakaryocyte development in cell culture.     

Factors required for bone marrow stromal fibroblast colony formation in vitro

Marrow stromal fibroblasts (MSFs) are essential for the formation of the haemopoietic microenvironment and bone; however, regulation of MSF proliferation is poorly understood. MSF colony formation was studied in primary mouse and human marrow cell cultures. After a brief exposure to serum, MSF colony formation occurred in the absence of both serum and non-adherent marrow cells, if medium conditioned by marrow cells was present (serum-free conditioned medium, SF-CM). In mouse and human cultures stimulated to proliferate by SF-CM, neutralizing antibodies against PDGF, TGF-β, bFGF and EGF specifically suppressed MSF colony formation. The degree of supression was species-dependent, with the most profound inhibition achieved in mouse cultures by anti-PDGF, anti-bFGF and anti-EGF, and in human cultures by anti-PDGF and anti-TGF-β. Serum-free medium not conditioned by marrow cells (SFM) did not support MSF colony formation. In mouse cultures in SFM, human recombinant bFGF and bovine natural bFGF were able to partially substitute for the stimulating effect of SF-CM. Other growth factors, including TGF-β₁, TGF-β₂, PDGF, EGF, IL-6, IGF-I and IGF-II, showed no activity when tested alone. In human cultures in SFM, none of the growth factors, alone or in combination, stimulated MSF colony formation. Mouse and human MSFs grown in SF-CM formed bone and a haemopoietic microenvironment when transplantated into immunodeficient mice in vivo , and therefore were functionally equivalent to MSFs generated in the presence of serum. These data indicate that stimulation of the initial proliferation of an MSF precursor cell is complex, and requires participation of at least four growth factors: PDGF, bFGF, TGF-β and EGF. In addition, mouse and human MSF precursor cells have different requirements for each of the growth factors.