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P Ralph

Publications and source records attributed to P Ralph.

At least 55 records · Page 3Linked to original sources
PubMed1986

Expression of the macrophage specific colony-stimulating factor (CSF-1) during human monocytic differentiation.

We and others have previously demonstrated expression of the co-fms proto-oncogene during human monocytic differentiation. The c-fms gene has since been shown to encode for the macrophage specific colony stimulating factor (CSF-1) receptor. The present results demonstrate that both CSF-1 and c-fms transcripts are induced during monocytic differentiation of human HL-60 leukemia cells. The results further demonstrate that normal human monocytes express CSF-1 RNA and that the level of these transcripts increases upon treatment with phorbol ester. Finally, the detection of CSF-1 RNA in HL-60 cells and in monocytes is associated with production of the CSF-1 gene product. These findings would suggest that monocytes are capable of regulating their own survival, growth and differentiation through CSF-1 production.

Cell Differentiation↗
PubMed1986

Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity.

CSF-1, a macrophage colony stimulating factor that causes proliferation and differentiation of progenitor cells, may also have effects on mature cells. Human peripheral blood monocytes were used to examine this possibility. Monocytes, separated from normal blood by density centrifugation and adherence, were incubated for 3 days with or without CSF-1 (1,000 U/ml, purified from the MIA PaCa pancreatic carcinoma line). The two groups of cells were then washed and tested for the ability, when induced, to produce several factors. When induced for 2 days with LPS and PMA, the monocytes produced a factor that was cytotoxic to L929 cells, and this factor was completely neutralized by polyclonal antibody to tumor necrosis factor. The cells preincubated with CSF-1 consistently produced an average of 12 times more of this factor than cells not exposed to CSF-1. Monocytes induced with LPS and PMA also produced a colony stimulating activity, as measured by colony formation when using mouse bone marrow. Cells preincubated with CSF-1, washed, and induced with LPS and PMA produced more than three times as much activity compared with control monocytes. When monocytes were induced with poly-I.C, 22-fold higher levels of interferon were produced by the cells exposed to CSF-1. These results show that CSF-1 has direct stimulating effects on mature human monocytes, and suggest that the macrophage growth factor may have clinical application in the treatment of infectious diseases and cancer.

Colony-Stimulating Factors↗
PubMed1986

Independent regulation of B-cell inducing factor and IL-2 production by T lymphocytes, and direct and indirect promotion of immunoglobulin secretion by glucocorticosteroid.

The conditions for induction of B-cell inducing factor (BIF) by human peripheral blood T cells was investigated. BIF was assayed by induction of immunoglobulin secreting cells (ISC) in peripheral blood B (non-T) cells stimulated with Staphylococcus aureus bacteria strain Cowan I (Sac), and in the IgM cell line SKW6.4. Maximum BIF production occurred with high concentrations of the T-cell mitogens phytohemagglutinin, concanavalin A, and PWM. Dexamethasone (Dex) also induced BIF production in T cells at 10(-5) to 10(-7) M. At 10(-5) and 10(-6) M Dex, the T-cell supernatants had to be dialyzed before testing because Dex alone stimulated variable levels of ISC in both test B-cell assays. Dex did not enhance BIF production by T cells that were optimally stimulated by lectin. BIF levels were maximum by Day 2 of T-cell cultures and remained high at Days 3 and 4. In contrast, IL-2 reached a peak at Day 1 and declined drastically by Day 4. We previously showed that IL-2 at less than 100 U/ml did not induce ISC in B cells and did not alter ISC induction by BIF. Dex did not induce IL-2 production and inhibited IL-2 production induced by Con A, in contrast to the promoting effects of Dex on BIF production, providing further evidence for the independence of BIF and IL-2 production and B-cell stimulation.

Adult↗
PubMed1986

Biological properties and molecular biology of the human macrophage growth factor, CSF-1.

CSF-1 is a growth and differentiation factor for the production of mononuclear phagocytes from undifferentiated bone marrow progenitors. In addition to previously described effects on mature cells, we show here that CSF-1 stimulates the production by monocytes of interferon, tumor necrosis factor, and myeloid CSF that produces mainly mixed neutrophil-macrophage colonies in bone marrow culture. Pretreatment with CSF-1 also promotes resistance to viral infection and tumor cytotoxicity in murine peritoneal macrophages. Based on amino acid sequence data of purified human urinary and murine L cell CSF-1, we have cloned the complementary DNA (cDNA) from messenger RNA (mRNA) of the human CSF-1 producing MIA PaCa cell line. The cDNA specifies a 32 amino acid signal peptide followed by a protein of 224 amino acids. Several facts suggest, however, that one-third of the molecule at the C-terminal end is processed off intracellularly to derive the secreted growth factor. The gene is about 18 kilobases (kb) in length and contains 9 exons. Although there appears to be a single copy gene for CSF-1, cells expressing the factor contain several mRNA species, suggesting that the gene may have several functions or levels of regulation. High level expression of the recombinant protein will allow preclinical testing in several disease models for therapeutic efficacy that has been suggested from in vitro and in vivo biological properties of CSF-1.

Animals↗
PubMed1986

Inducible production of human macrophage growth factor, CSF-1.

A panel of human cell lines was screened for production of colony-stimulating factor-1 (CSF-1) using a specific radioreceptor assay and criterion of macrophage colony growth in mouse bone marrow culture. The pancreatic carcinoma lines MIA PaCa and PANC were found to secrete high levels of CSF-1. In a bone marrow proliferation assay, the activities from these two lines were blocked by a CSF-1 specific neutralizing antiserum, confirming the predominant content of this macrophage growth factor. MIA PaCA cells stopped secreting CSF-1 when transferred to various serum-free media. Serum-free production could be reinitiated by phorbol myristic acetate (PMA). Purified CSF-1 from serum-free MIA PaCa cells stimulated the formation of 14-day colonies from total and nonadherent mononuclear human bone marrow cells. Most of the colonies consisted exclusively of large, dispersed macrophages that were intensely stained for nonspecific esterase. Although similar numbers of human 14-day colonies were stimulated by CSF-1 and other CSFs, more CSF-1 was required for the proliferation of human as compared with murine bone marrow progenitors. Northern analysis of mRNA from induced-MIA PaCa cells, using a human CSF-1 oligonucleotide probe, revealed multiple species of CSF-1 mRNA ranging from 1.5 to 4.5 kilobases (kb). Uninduced, serum-free cultures showed only the largest mRNA species, suggesting that serum removal interfered with CSF-1 mRNA processing related to synthesis and/or secretion of the protein. Regulation of the production of CSF-1 may be an important physiological process in hematopoiesis and macrophage functioning.

Animals↗
PubMed1985

Biochemical characterization and purification of human B cell stimulatory factor (BSF).

B cell stimulatory factor (BSF) activity was generated over a period of five days by phytohemagglutinin-stimulated E rosette-positive peripheral blood lymphocytes. This activity was subjected to a multistep purification procedure including ammonium sulfate precipitation, anion-exchange chromatography, gel filtration, procion red-agarose chromatography and reverse phase high performance liquid chromatography (RP-HPLC). The last purification step dissected BSF activity into two active fractions, one corresponded to the interleukin 2 (IL 2) activity whereas the other active fraction was free of IL 2 activity. Preparative isoelectric focusing analysis defined isoelectric points of pH 7.2 for both BSF and IL 2. Molecular weight analysis of BSF was carried out by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. BSF activity was eluted from gel strips corresponding to a molecular mass of 16 and 17 kDa. The 16-kDa fraction was free of IL 2 activity whereas the 17-kDa fraction overlapped with IL 2. Since recombinant IL 2 was capable of exhibiting significant BSF activity in anti-IgM or Staphylococcus aureus Cowan strain I-dependent assay systems, it cannot be excluded that IL 2 itself is a BSF. Nevertheless these studies demonstrate the existence of a BSF free of IL 2 activity.

Antibodies, Anti-Idiotypic↗
PubMed1985

Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines.

The human leukemic myeloblast HL-60 and monoblast U937 cell lines have made important contributions to the disciplines of cancer, hematology, and immunology. As sources of leukemic cells, they have been used for the study of neoplasia and therapeutics. As sources of hemic cells, they have been used for biochemical and biological analysis of regulation and differentiation in myelopoiesis. When stimulated with immunomodulatory factors, the cell lines develop properties of host-defense effector cells. They are also a source of cytokines that affect other cell types.

Cell Differentiation↗
PubMed1985

Rescue of IgM, IgG, and IgA production in common varied immunodeficiency by T cell-independent stimulation with Epstein-Barr virus.

We previously defined three categories of B-cell defects in common varied immunodeficiency (CVI): failure to produce IgG and IgA in response to T cell-dependent (TD) stimulation by Staphylococcus bacteria (Sac) plus pokeweed mitogen or B-cell inducing factor (BIF), failure to produce any immunoglobulin, and failure of Sac-induced proliferation and differentiation. The present study includes the responses of 22 CVI patients to T cell-independent (TI) stimulation by Epstein-Barr virus (EBV). In the majority of patients, EBV-stimulated B cells showed normal proliferation and IgM production. In addition, IgG and IgA production was in the range of that for EBV-stimulated normal cells in many patients. Among 11 patients with no TD production of immunoglobulin of any isotype, two showed normal IgM secretion in response to EBV and five others had significant but subnormal responses. Four patients never had humoral responses despite repeated testing and removal of potentially suppressing T cells and monocytes. Concanavalin A stimulation of the T cells from all the patients tested resulted in the production of B-cell inducing factor at higher levels than for normal donor T cells, as assayed on normal Sac-stimulated B cells. These results show that many cases of B-cell defects in CVI patients involving TD production of IgM, switching to TD production of IgG and IgA, and mitogen responses to Sac are not absolute defects. The B cells will respond normally to some stimuli.

Adolescent↗
PubMed1985

Distinct activities of interferon-gamma, lymphokine and cytokine differentiation-inducing factors acting on the human monoblastic leukemia cell line U937.

The human monoblast leukemia line U937 is growth inhibited and induced to express various characteristics of mature monocytes by lymphokines (LK) and other cytokines. Previous experiments have shown that interferon-gamma (IFN-gamma) is responsible for some but not all of the differentiation-inducing effects on U937. To determine the variety and specificity of activity, the following factors were studied: phytohemagglutinin-induced LK that contained IFN-gamma (100 units/ml); purified IFN-gamma; human colony-stimulating factor 1 (CSF-1); and conditioned medium(a) (CM) from the human bladder carcinoma cell line 5637 and the hepatoma cell line SK-HEP. LK preparations contained no colony-stimulating activity, whereas CM from 5637 and SK-HEP both contained granulocyte-macrophage CSF (3000 to 4000 units/ml) but no IFN activity. IFN-gamma is the major immunoglobulin G Fc receptor-inducing species within lymphokine, since anti-interferon-gamma antibody inhibited most of this activity. Other sources of Fc receptor-inducing activity were CM from SK-HEP and 5637 cell lines. Human CSF-1 when tested up to 800 units/ml was inactive for Fc receptor induction. LK induced the chemotactic peptide receptor, but this induction was due to factors other than IFN-gamma as anti-IFN-gamma antibody did not inhibit the induction, and purified IFN-gamma at a dose equivalent to that found in LK (100 units/ml) had no activity in the assay. SK-HEP and 5637 CM had strong chemotactic peptide receptor-inducing activity, but human CSF-1 was inactive up to 800 units/ml. Peroxide production after stimulation with phorbol myristic acid could be induced by LK, LK with anti-IFN-gamma antibody, 5637, and SK-HEP treatment. IFN-gamma (100 units/ml) and CSF-1 (800 units/ml) were ineffective. Peroxide production was induced by IFN-gamma at concentrations above 1000 units/ml. The inducibility of several enzymatic activities was determined as additional measures of maturation. N-Acetylglucuronidase was induced, for example, by LK, IFN-gamma, 5637 CM, and phorbol myristic acid. Alkaline phosphatase was induced by LK, IFN-gamma, dexamethasone, and phorbol myristic acid. 1,25-Dihydroxycholecalciferol was also examined and could induce most of the maturational markers examined. The results demonstrate that non-IFN cytokines from several sources have profound differentiation-inducing effects on monoblast leukemia cells in a pattern different from that of IFN-gamma.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗
PubMed1985

Distinct differentiation-inducing activities of gamma-interferon and cytokine factors acting on the human promyelocytic leukemia cell line HL-60.

The human promyelocytic leukemia cell line HL-60 and monoblastic leukemia cell line U937 undergo differentiation when induced by lymphokine and cytokine preparations. Growth inhibition, acquisition of immunoglobulin Fc receptors, increased expression of monocyte-related surface antigens, and an increase in lysosomal enzyme contents accompany maturation induced by gamma-interferon and other cytokine factors tested. Additionally, increased receptors for chemotactic peptide (fMLPR), increased hydrogen peroxide release in response to phorbol myristic acetate stimulation, and the release of prostaglandins (PGE2 and 6-keto-PGF1a) follow exposure to lymphokine and cell line sources of myeloid colony-stimulating activity (CSA). Gamma-Interferon (gamma-IFN) induced fMLPR in HL-60 (only at 1000 units/ml) but not in U937. Additionally, gamma-IFN did not induce prostaglandin release in either cell line. These myeloid colony-stimulating activity-associated differentiation-inducing factors were obtained from the human hepatoma++ cell line SK-Hep and bladder carcinoma cell line 5637, which were free of interferon activity. The 2-day phytohemagglutinin-induced lymphokine contained no detectable CSA and was a good source of differentiation activity. A simple, rapid assay for a new human CSA with pluripotent hematopoietic stimulating activity (pluripoietin) is described based on stimulation of [3H]glucosamine incorporation. Cell line conditioned media containing pluripoietin, purified pluripoietin, and gamma-IFN are active in this assay. These myeloid leukemia cell line differentiation factors are thus different from interferon and conventional CSA. These results suggest that endogenous human cytokines may have a role in the differentiation of leukemic as well as normal myeloid cells.

Antibodies, Monoclonal↗
PubMed1984

Cooperation of IgG monoclonal antibodies in macrophage antibody-dependent cellular cytotoxicity (ADCC) to tumor targets.

Six IgG monoclonal antibodies representing the four murine IgG isotypes were active individually in ADCC to T-lymphoma targets mediated by murine macrophages and human blood K cells. The monoclonals were directed against four antigens (Thy-1.2, H-2k, Ly 2.1, Ly 9.2). None was as effective in ADCC as allo-anti-Thy-1.2 serum or rabbit anti-mouse spleen serum, even at plateau levels of killing. Monoclonals gave the highest levels of ADCC at relatively low amounts of antibody bound to targets; increasing the amount of bound antibody by 10- to 100-fold did not increase murine macrophage-mediated cytotoxicity. In contrast, ADCC using alloantiserum continued to increase over the same range of antibody bound to the tumor targets. The activity of individual monoclonals was not enhanced by the presence of various dilutions of normal mouse or rabbit serum, suggesting that the superiority of the allo- and hetero-antisera was due to their content of heterogeneous antibodies. IgG monoclonals of different isotypes and recognizing different antigens gave enhanced ADCC in combination; monoclonals to the same antigen did not. An IgM anti-Thy-1.2 monoclonal was inactive in ADCC and inhibited the activity of IgG monoclonals of the same specificity. These studies show that IgG antibodies of different specificity and class can collaborate in ADCC, and that this cooperative effect is not due simply to increased amounts of antibody bound to the tumor targets.

Animals↗
PubMed1984

Interleukin-2 correction of defective in vitro T-cell mitogenesis in patients with common varied immunodeficiency.

We studied the ability of phytohemagglutinin (PHA) and two anti-T-cell monoclonal antibodies, OKT3 and Pan T2, to induce interleukin-2 (IL2) production and proliferation in peripheral blood lymphocytes (PBL) from 14 patients with combined varied immunodeficiency (CVI). The median values of endogenous IL2 produced by mitogen-stimulated PBL was significantly lower in patients than controls irrespective of the mitogen used. The patients, taken as a group, had a significantly decreased in vitro PBL response to mitogen stimulation when compared to controls. With the addition of a highly purified human IL2 preparation, the proliferative response in the majority of patients was significantly improved with all mitogens. Three patient groups could be distinguished: Group A (3/14) had full restoration of proliferative response with the addition of IL2, Group B (5/14) had partial restoration, and Group C (6/14) had no significant response. The monoclonal antibody, Pan T2, recognized a T-cell proliferative defect in 5 of 14 patients which neither PHA nor OKT3 recognized. This was not significantly corrected by the addition of IL2. This T-cell proliferative defect correlated with the lack of B-cell proliferation and immunoglobulin production in response to B-cell mitogens in three-fourths of the patients assayed. These data show that CVI patients are a heterogeneous group but have in common a decreased in vitro production of IL2 resulting in a proliferative defect which is correctable at least in part, in vitro, in the majority by the addition of purified IL2.

Adolescent↗
PubMed1984

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2.

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.

Animals↗
PubMed1984

Human B cell-inducing factor(s) for production of IgM, IgG and IgA: independence from IL 2.

Human peripheral blood B cells are stimulated into proliferation by killed Staphylococcus aureus bacteria strain Cowan I (Sac). T lymphocytes in the presence of a T cell mitogen induce high numbers of immunoglobulin-secreting cells (ISC) in these Sac-stimulated B cells. The T cells can be largely replaced by a lymphokine factor. We describe here the 11000-fold purification of this B cell-inducing factor (BIF). BIF preparations that are free of IL 2 do not require IL 2 for optimal induction of ISC. This was shown by the lack of effect of IL 2 alone or with suboptimal or optimal concentrations of BIF on the induction of ISC and by the absence of IL 2 production in the purified B cell population which, with other controls, excludes significant T cell contamination. BIF, purified through four fractionation steps and free of IL 2, induces IgM, IgG, and IgA-ISC in approximately the same ratio as unfractionated lymphokine. Because we have not yet attained a pure BIF preparation, the possibility of separate factors for the production of each immunoglobulin isotype cannot be ruled out.

Adult↗
PubMed1983

Lymphokine factors inducing IgG production in human B-cell line ARH-77 and stimulatory effects of phorbol ester tumor promoter.

Regulation of immunoglobulin-secreting cells (ISC) was studied in human IgG B-cell line ARH-77, as assayed by reverse plaque formation. Numbers of ISC were stimulated by both lymphokine (stimulation index = 1.5-8.7) and phorbol myristic acetate (PMA) (stimulation index = 2.7-42). Incubation of ARH-77 with the two agents together caused additive or super-additive numbers of ISC, suggesting that they acted on this B cell via different mechanisms. B-cell-inducing factors stimulating IgG ISC in ARH-77 line were found at 20,000 and 40-60,000 molecular mass. The 20-KDa factor could be distinguished from IL-2 by affinity chromatography on blue agarose. Stimulated cells maintained their immunoglobulin class, and no evidence for isotype switching to IgM or IgA was detected using lymphokine or PMA. The cell line is a model for normal B lymphocytes which have been activated, for example, by Staphylococcus bacteria, to respond to T-cell factors.

Antibody-Producing Cells↗