Development of natural killer cells, B lymphocytes, macrophages, and mast cells from single hematopoietic progenitors in culture of murine fetal liver cells

We have recently established a clonal culture system that supports the growth of immature natural killer (NK) cells from murine fetal thymocytes. We now describe a culture system for mixed NK cell colony formation from single lymphohematopoietic progenitors. When Sca-1+c-kit+ fetal liver cells were cultured in methylcellulose media with interleukin (IL)-2, IL-7, IL-11, and steel factor (SF), we found mixed colonies consisting of diffuse small round cells characteristic of immature NK cells and other types of cells. The single cell origin of the mixed colonies was established by micromanipulation. Individual mixed colonies derived from single cells were characterized by flow cytometric analysis and May-Grünwald Giemsa staining. All mixed colonies contained Thy-1+B220- cells, which can differentiate to mature NK cells in fetal thymus organ culture. Most of the colonies contained B220+ B-lineage cells and macrophages, and some contained mast cells. IL-1alpha and IL-3, which have previously been shown to inhibit the T- and B-cell potentials of blast colonies, suppressed the formation of mixed NK cell colonies. The clonal culture assay presented here may be useful in analysis of the developmental pathway and commitment of NK cells from multipotential progenitors.     

Cytokine receptors, growth factors and cell cycle in human bone marrow and peripheral blood hematopoietic progenitors

BACKGROUND AND OBJECTIVE: An increasing number of growth factors have been shown to be responsible for the proliferation, survival and enhanced function of many cell types within the hemopoietic system. The action of these hemopoietic growth factors in stimulating cell growth and survival applies both to cells within the progenitor compartment and mature cells. Whether a specific cytokine influences in vivo hematopoietic progenitor cell proliferation or survival depends on cytokine-mediated modulation or target cell cytokine receptors, cell proliferation, and cell death regulator genes and other pathways. To address these issues, particularly in view of the current and future clinical use of hemopoietic growth factors, the Italian Society of Experimental Hematology organized a Meeting in Florence on July 4th, 1996. INFORMATION SOURCES: The material examined in the present review includes full papers and abstracts published in journals covered by the Science Citation Index and Medline. All the participants to the Meeting in Florence have been actively working in the field of biology and clinical application of hemopoietic growth factors. Summaries of their oral presentations at the Florence Meeting are reported in the Appendix to this article. STATE OF ART AND PERSPECTIVES: Myelopoietic growth factors particularly granulocyte (G-) colony-stimulating factor (CSF) and granulocyte-macrophage (GM)-CSF, have been available for clinical use for only a few years but they have already markedly changed the management of chemotherapy-induced neutropenia, the use of dose-intensive chemotherapy regimens and the practice and safety of autologous stem cell transplantation. While these growth factors have been rapidly introduced as routine agents in the management of cancer patients, they have continued to generate a considerable amount of fundamental research into the biology of hematopoiesis as well as the growth regulation of normal and cancer cells. For instance, one goal of cancer treatment is to protect hematopoietic stem and progenitor cells from the damaging effects of chemotherapy, while maintaining their anticancer action. Any means of preferentially and reversibly suppressing the proliferation of normal hematopoietic stem and progenitor cells while leaving the proliferation of tumor cells and their susceptibility to chemotherapy unmodified, could potentially optimize treatment efficacy. In this field, the possibility of using colony-stimulating factors as myeloprotective agents in dose-intensive chemotherapy to enhance anticancer activity could be an attractive goal of current anti-cancer treatment modalities.     

Costimulation, tolerance and ignorance of cytolytic T lymphocytes in immune responses to tumor antigens

Despite the fact that many tumors express MHC class I molecules presenting "foreign" peptide antigens, a vigorous tumor-destructing immune response is seldom detected. A possible explanation is that tumors cannot provide adequate costimulatory signals as provided by professional antigen presenting cells. CD28, upon interacting with B7, triggers costimulatory signals critical for the T-cell response. Transfection of tumor cells with B7 augments the immunogenicity of the tumor so that an anti-tumor immune response can be amplified. When B7-CD28 costimulation is provided CTL specific for otherwise silent epitopes can be activated. Therefore, unresponsiveness of T cells to many tumor antigens should be considered as ignorance rather than tolerance. Immunological ignorance may thus contribute to the failure of the immune system to respond against the tumor antigens.     

c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy

The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, several dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions.     

Cytotoxic lymphocytes in the peripheral blood of patients with B cell lymphomas

In order to analyze systemic immune surveillance in patients with B cell non-Hodgkin's lymphomas (B-NHL), we investigated circulating lymphocytes using two-color flow cytometry. The proportions of CD3-CD56+ natural killer (NK) cells and CD8++(bright) S6F1++ killer-effector T cells corresponding to activated cytotoxic T lymphocytes (aCTL) were studied in the peripheral blood of 26 patients with indolent lymphoma (IL) and 24 with aggressive lymphoma (AL). The AL patients with both limited disease and advanced disease had an increased proportion of NK cells. However, this feature was not evident in IL patients with either limited or advanced disease. In contrast, an increased proportion of aCTL was observed only in IL patients with advanced disease. These findings indicate that IL may differ from AL in terms of immune surveillance against neoplastic B cells.     

Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors

Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokines, is secreted by interferon gamma-stimulated T cells, monocytes, endothelial cells, and keratinocytes. We have begun to explore the biological properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was secreted by infected insect cells, which on sodium dodecyl sulfate-polyacrilamide gel electrophoresis comigrated with keratinocyte IP-10 and with f(22-98), a bacterial recombinant fragment lacking the signal sequence but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alpha IP-10) and 77-98 of IP-10 (alpha 22), demonstrating that it is secreted by keratinocytes and insect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP-10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-steel factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations > or = 50 ng/ml, is prevented by preincubation of rIP-10 with alpha IP-10, but not by alpha 22, and is seen with highly purified CD34+ cells, suggesting direct effect of rIP-10 on the progenitors. Combination of rIP-10 and other chemokines at inactive concentrations inhibited colony formation in a synergistic manner. rIP-10 did not affect colony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemotherapy.     

Bone marrow clones representing an intermediate stage of development between hematopoietic stem cells and pro-T-lymphocyte or pro-B-lymphocyte progenitors

We have established in culture several nontransformed bone marrow clones (called PR) that show phenotypic and genotypic characteristics that distinguish them from totipotent stem cells and lineage-restricted Pro-T lymphocytes, Pro-B lymphocytes, and myeloid cell progenitors. In vivo and/or in vitro the PR clones give rise to T lymphocytes, B lymphocytes, and some myeloid-lineage cells, but they appear not to be able to generate cells of the erythroid lineage, nor can they rescue mice from a lethal dose of irradiation. We conclude that the PR clones are precursor cells representing an intermediate stage of development between the totipotential stem cell and lineage-restricted progenitor cells. The results described here support a model of blood cell formation in which stem cell differentiation is a progressive process marked by the stepwise loss of self renewal and functional potential. In addition, they provide evidence that cytokines and specialized microenvironments can direct the fate of the developing multipotent progenitor cells.     

Effect of nicotine on the immune system: possible regulation of immune responses by central and peripheral mechanisms

Nicotine (NT) treatment impairs T-cell receptor (TCR)-mediated signaling, leading to the arrest of T cells in the G1 phase of the cell cycle and inhibition of the antibody plaque-forming cell (AFC) response to sheep red blood cells (SRBC). This paper summarizes some of the previous findings related to cigarette smoke/NT and the immune response, and presents preliminary evidence suggesting that mice chronically treated with NT (0.5 mg/day/kg body weight) have a depressed inflammatory response in the turpentine-induced abscess model of inflammation. This ability of nicotine to attenuate an inflammatory response may also be the cause of reduced mortality of chronically nicotine-treated mice from acute influenza A pneumonitis. Moreover, in LEW rats, decreased anti-SRBC AFC responses were also observed after intracerebroventricular (i.c.v.) administration of relatively small concentrations of NT (28 micrograms/day/kg body weight) which, when given peripherally, did not affect the AFC response. In vitro the addition of NT to T cells increased protein tyrosine kinase (PTK) activity and intracellular Ca2+ concentration [Ca2+]i. These results support the hypothesis that NT alters immune responses by directly interacting with T cells, as well as indirectly through brain-immune interactions.     

Relative sedimentation of hematopoietic progenitors in human cord blood, peripheral blood, and bone marrow as determined by counterflow centrifugal elutriation

OBJECTIVE: Although there have been many studies of the outcome of anorexia nervosa, methodological weaknesses limit their interpretation. The authors used a case-control design to try to improve knowledge about the outcome of anorexia nervosa. METHOD: All new female patients referred to an eating disorders service between Jan. 1, 1981, and Dec. 31, 1984, who had probable or definite anorexia nervosa were eligible for inclusion. Of these women, 86.4% (N = 70) were located and agreed to participate. The comparison group (N = 98) was a random community sample. All subjects were interviewed with a structured diagnostic instrument. RESULTS: A minority of the patients (10%) continued to meet the criteria for anorexia nervosa a mean of 12 years after initial referral. Even among those who no longer met these criteria, relatively low body weight and cognitive features characteristic of anorexia nervosa (perfectionism and cognitive restraint) persisted. The rates of lifetime comorbid major depression, alcohol dependence, and a number of anxiety disorders were very high. CONCLUSIONS: In the managed care/brief treatment era, therapeutic approaches with an excessive focus on weight gain that neglect the detection and treatment of associated psychological features and comorbidity may be inappropriate. Anorexia nervosa is a serious psychiatric disorder with substantial morbidity.     

Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.     

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.     

Decreased expression of LFA-1 on peripheral blood lymphocytes in graft versus host disease

We have previously demonstrated an increase in lymphocyte function associated antigen-1 (LFA-1) expression in the native intestines of animals with graft versus host disease (GVHD) after small bowel transplantation (SBTx). The present study evaluated LFA-1 expression on peripheral blood lymphocytes (PBLs) during GVHD. GVHD was created in LBNF1 rats by heterotopic vascularized SBTx from Lewis donors and compared to sham-op controls and cyclosporine-treated SBTx rats (SBTx-CyA, 10 mg/kg/day). PBLs were harvested on Postop Day 13 when signs of severe GVHD were present and PBLs were stained for LFA-1, CD3 (T-cell marker), and CD45 (B cell marker) and analyzed on an Epics 5 flow cytometer. Mesenteric lymph node (MLN) lymphocytes from the native intestines were also harvested for each group and stained for LFA-1. There were significant decreases in LFA-1, CD3, and CD45 PBL expression in rats with GVHD. CyA treatment corrected the abnormal CD3 and CD45 ratios, but not LFA-1 expression. It is concluded that: (1) The proportion of PBLs expressing LFA-1 is depressed in animals with clinical GVHD consistent with their recruitment into the host's inflamed tissues. (2) CyA treatment corrects the abnormalities in T and B cell ratios but not the decreased expression of LFA-1 on PBLs and may relate to its known imperfect ability to treat GVHD.     

Different protein kinase C isoenzymes regulate IL-2 receptor expression or IL-2 synthesis in human lymphocytes stimulated via the TCR

Stimulation of purified human PBL with mAbs raised against the T cell receptor resulted in an immediate and transient activation of protein kinase C-alpha (PKC-alpha) and PKC-theta, peaking at 10 min, whereas PKC-beta, -delta, and -epsilon were translocated with a delay of >90 min and remained activated for up to 2 h. To characterize specific functions of distinct PKC isoenzymes, Abs against different PKC isoenzymes were introduced by means of electropermeabilization. Neutralization of PKC-alpha and -theta resulted in the complete inhibition of IL-2R expression, whereas anti-PKC-beta, -delta, and -epsilon Abs inhibited IL-2 synthesis. Extensive control experiments have shown that neither electropermeabilization nor control Ig influenced PKC activity and cellular functions. Our data thus clearly show that specific PKC isoenzymes regulate different cellular functions in stimulated human lymphocytes.     

Efficient peripheral clonal elimination of B lymphocytes in MRL/lpr mice bearing autoantibody transgenes

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2(d) genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83mu delta, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2(d) mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2(d) mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2(d) mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2(d) mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2(d) mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2(d) mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.     

RhG-CSF-mobilized CD34+ peripheral blood progenitors are myeloperoxidase-negative and noncycling irrespective of CD33 or CD13 coexpression

OBJECTIVE: The purpose of this study was to differentiate between the catabolic effects of interleukin-1 (IL-1) when infused into the central nervous system or into the periphery. SUMMARY BACKGROUND DATA: After injury and infection, IL-1 has been found in the central nervous system. Chronic intracerebrovascular infusion of IL-1 produces protein catabolism, anorexia, and fever. However, IL-1 may act directly on liver and bone marrow to elicit metabolic responses. Although IL-1 is thought to be involved in a number of metabolic responses associated with injury-inflammation, the sites of action are unclear. METHODS: Rats were implanted with chronic infusion pumps and received diluent or three doses of IL-1 infused subcutaneously for 6 days, and a variety of response variables were measured. In a second study, doses were adjusted so that similar systemic catabolic responses were obtained from peripheral and intracerebroventricular infusion of IL-1. The acute-phase responses then were compared in the two groups of animals receiving IL-1 by different routes. RESULT: Subcutaneously infused IL-1 elicited catabolic responses in a dose-response manner. Similar catabolic responses were achieved by infusing one tenth of the dose of IL-1 given subcutaneously into the central nervous system. Although similar systemic responses (protein catabolism, anorexia, fever, and weight loss) were observed with both routes of infusion, the subcutaneous infusion produced a much greater lymphocytosis, elevation in acute-phase reactants, and fall in serum iron and albumin. CONCLUSIONS: The IL-1 appears to have different effects depending on the site of production and site of action. Regionalization of signal proteins such as IL-1 should be taken into consideration when devising specific anticytokine treatment strategies.     

Ro/SS-A-reactive B lymphocytes in salivary glands and peripheral blood of patients with Sj?gren's syndrome

The aim of this study was to investigate the production of anti-Ro/SS-A antibodies in labial salivary glands (LSG) and peripheral blood (PB) of Sj?gren's syndrome (SS) patients. The ELISPOT method was performed to quantify the frequency of LSG lymphocytes and PB lymphocytes spontaneously secreting anti-Ro/SS-A antibodies. The total number of IgG-, IgA- and IgM-producing cells was also quantified. The bovine Ro 60-kD protein was used as target antigen. Six of six primary SS patients had LSG B cells producing anti-bovine Ro 60 kD of the IgG isotype, and two of two primary SS patients had in addition PB lymphocytes producing anti-bovine Ro 60 kD of the IgG isotype. The six patients who had IgG antibodies against the Ro/SS-A antigen in LSG all had focus scores of >/= 7 in biopsies of LSG. The results indicate that SS patients with a high degree of local inflammation in LSG have B cells producing anti-Ro/SS-A antibodies in both LSG and PB. Thus, the anti-Ro/SS-A antibodies may have pathogenic importance in the progression of the exocrinopathy of SS.