Overproduction of PGE2 in peripheral blood monocytes of gastrointestinal cancer patients with mucins in their bloodstream

When monocytes from healthy donors were cultured in the presence of sera from patients with gastrointestinal cancer, PGE2 production from the monocytes was elevated. Serum proteins were fractionated on Sepharose 4B and the inducing activity was found in the excluded fractions. By excluding some mucins from the serum, the inducing activity was reduced effectively. The activity was also reduced by adding binding inhibitors to the scavenger receptor. These results suggest that peripheral blood monocytes in epithelial cancer patients may be continuously stimulated by mucins in the bloodstream through the scavenger receptor, resulting in overproduction of PGE2.     

Heterogeneity of human lymphokine (IL-2)-activated killer (LAK) precursors and regulation of their LAK induction by blood monocytes

Highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated by CCE from peripheral blood of healthy donors. Blood lymphocytes were separated by this CCE into 9 subpopulations. The NK activities of these lymphocyte fractions against NK-sensitive K-562 cells and their LAK activities against NK cell-resistant target (Daudi) cells were assayed promptly or after incubation of the fractions for 4 days with or without an optimal concentration of IL-2. NK and LAK activities were measured by 4-hr 51Cr-release assay. On the basis of their NK and LAK activities, these lymphocyte fractions were classified into 3 subpopulations of LAK precursors: one lacking both NK and LAK activities (Fr.2), one with moderate NK activity but low LAK activity (Fr.5), and one possessing both NK and LAK activities (Fr.8). Addition of autologous fresh monocytes to the lymphocyte cultures resulted in a significant increase in induction of LAK activity in Fr.2 and Fr.5. This up-regulation of lymphocytes in Fr. 2 and Fr.5 by monocytes was confirmed in parallel experiments by measuring the blastogenic response of the lymphocytes to IL-2. Deletion of lymphocytes in Fr. 8 of CD16+ (Leu-11+) NK cells resulted in 74% reduction in LAK induction, whereas depletion of mixtures of monocytes and lymphocytes in Fr. 2 of cells reacting with CD3+ (OKT3+) antibody resulted in a 66% reduction in LAK induction. This up-regulation of LAK cell induction from LAK precursors by monocytes was confirmed using 4 lines of human lung cancer cells as targets for LAK activity. These results clearly indicate that human monocytes may cause up-regulation of the expression of IL-2-induced LAK activity in T cells and in a subpopulation of NK cells.     

Defective activity of monocytes from patients with non-Hodgkin lymphoma. The modulatory effect of granulocyte-macrophage-colony stimulating factor

BACKGROUND: Mononuclear phagocytic function is not well defined in non-Hodgkin lymphoma patients although, defective function of those cells has been reported in patients with Hodgkin disease and other solid tumors. The potential application of granulocyte-macrophage-colony stimulating factor (GM-CSF) in the prevention and treatment of infections in those patients is being studied. METHODS: Phagocytosis and microbiocidal activity of monocytes in peripheral blood from 10 newly diagnosed patients and 14 healthy donors were tested cytologically against a strain of Candida albicans, and chemotaxis was evaluated in a Boyden chamber using zymosan-activated serum as a chemotactic agent. Cells were assayed under basal conditions and after incubation with GM-CSF (12 ng/mL). RESULTS: The phagocytosis and chemotactic activity of monocytes from non-Hodgkin lymphoma patients was lower than results obtained with cells from healthy donors (P < 0.05), and microbiocidal activity against Candida albicanswas similar in both groups. After exposure to GM-CSF, the functional activity of monocytes from control donors was only slightly modified (P > 0.05); by contrast, the percentage of mononuclear phagocytic cells in non-Hodgkin lymphoma (NHL) patients increased from 41 +/- 3% to 53 +/- 3%, the phagocytic index from 0.6 +/- 0.1 to 0.87 +/- 0.1 (P < 0.05), microbiocidal activity against Candida from 54 +/- 5% to 66 +/- 6% (P > 0.05), and chemotaxis from 43 +/- 8 cells per field to 48 +/- 9 cells per field (P > 0.05). CONCLUSIONS: The results of this study indicate that there is defective phagocytic and chemotactic activity in monocytes from NHL patients at diagnosis. "In vitro" improvement of phagocytic activity was observed after exposure to GM-CSF.     

A unique monoclonal antibody against human plasma fibronectin, which recognizes an epitope on the surface of a subpopulation of polymorphonuclear neutrophils

A new monoclonal antibody (MAb) specific to plasma fibronectin, named MO, which was produced by immunizing a mouse with fragments of fibronectin isolated from human plasma, was found to bind to the surface of a subpopulation of peripheral blood polymorphonuclear neutrophils (PMNs) in the presence of physiological concentration of soluble fibronectin. MAb MO had no reactivity with other blood cells such as lymphocytes, monocytes, or platelets. The percentage of the MAb MO-reactive subpopulation of PMNs from healthy donors varied from 0 to 60%. Ten previously established MAbs against fibronectin tested in this study together with MAb MO had no reactivity with the surface of PMNs at all, when tested simultaneously with MAb MO. These results indicate that MAb MO can be a unique and useful tool for analyzing the structure and function of fibronectin-especially the fibronectin expressed on the surface of PMNs.     

Detection of both T-cell and Ia-like antigens on cells from patients with acute myelomonocytic leukemia and chronic myelogenous leukemia in blast crisis.

Appropriately absorbed antisera to the lymphoblastoid cell lines HSB and SB detect a human T-lymphocyte-associated antigen (TLAA) and the human Ia-like antigens, respectively. Cells from some patients with acute myelomonocytic leukemia (AMML) and chronic myelogenous leukemia in blast crisis expressed both TLAA and Ia antigens when tested in a complement-dependent microcytotoxicity assay (greater than 90% lysis with both antisera). When patients were in remission, expression of TLAA and Ia antigens returned to normal values. Quantitative absorption of anti-TLAA serum with increasing numbers of AMML cells showed that these cells could remove reactivity of the serum for both HSB and human thymocytes. Similarly, absorption of anti-Ia serum with AMML cells removed all serological reactivity when this serum was tested on chronic lymphocytic leukemia cells or normal B-cells. These serological findings were confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies using radiolabeled antigens. Cells from an AMML patient were labeled with 125I using lactoperoxidase; both the TLAA and Ia antigens were precipitated from the resulting solubilized membrane preparation. Leukemic cells from one AMML patient and one patient with chronic myelogenous leukemia in blast crisis were studied for Ia and TLAA antigens with a double fluorescence technique. Over 80% of the cells showed dual fluorescence.     

Separation of chronic lymphocytic-leukemia (cll) cells by a discontinuous density gradient - correlation with clinical stage

Peripheral blood or bone marrow cells of 15 patients with chronic lymphocytic leukemia (CLL) were separated on an albumin density gradient. In 5 of 5 CLL patients, the distribution of malignant B lymphocytes was similar across the gradient when blood and bone marrow were compared, but different from the distribution of cells from healthy volunteers. In 10 patients, B cell colony formation was measured in vitro from peripheral blood cells after gradient fractionation. Although most of the cells in all patients were found in fraction 4, the majority of B-lymphocyte colonies were found in light density fractions (1+2, 3) in patients with more advanced disease (Rai stages 3 or 4), and in heavier fractions (4, bottom) in patients with less advanced disease (Rai stages 0, 1 or 2). The density of CLL cells might provide a new prognostic marker in this disease.     

Induction of human monocyte-mediated tumor cell killing by a plant lectin, wheat germ agglutinin

Human peripheral blood monocytes from healthy donors were separated by discontinuous gradient centrifugation and adherence to yield highly purified adherent cell populations (greater than 99% monocytes). Five different plant lectins were tested for ability to induce lectin-dependent monocyte-mediated cytotoxicity (LDMC). Only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible LDMC activity. All the tumor target cells tested were sensitive to variable extents to cytotoxicity mediated by WGA-treated monocytes. Pretreatment of monocytes with WGA did not result in development of LDMC. N-Acetylglucosamine, which specifically binds WGA, inhibited WGA-dependent monocyte-mediated cytotoxicity. Treatment of adherent monocyte-rich monolayers with monoclonal anti-natural killer cell antibody (anti-Leu-11b) and complement did not affect the LDMC activity induced by WGA. These results indicate that the plant lectin WGA, which binds specifically to both human monocytes and tumor cells, renders human blood monocytes cytotoxic to human tumor cells.     

Mononuclear-cell infiltration in ovarian cancer. I. Inflammatory-cell infiltrates from tumour and ascites material.

Malignant effusions and tumour tissue obtained at surgery provided material for a study of the prognostic value of the various inflammatory cells in the prognosis of human ovarian cancer. Ascitic fluids predominantly contained inflammatory cells; tumour cells, both singly and in clusters, were a minor component. Tumour cells were usually in excess in dispersed solid material. Some patients had significant proportions of lymphocytes and macrophages in their solid tumour, and these patients invariably responded to therapy. Sedimentation-velocity separation at unit gravity provided tow populations of inflammatory cells. One consisted of mononuclear cells similar in size to those in the patients blood: the other consisted of one or more large macrophage populations, distinct in morphology and enzymatic markers from both blood monocytes and each other. T lymphocytes were enriched in ascites fractions (78%) and in the tumour-derived mononuclear fraction (71%) compared to patient blood (60%). The T-cell subset characterized by ANAE reactivity was markedly depleted in the tumour-infiltrating fraction (17%) compared to patient blood (62%) or patient blood (51%). Esterase-positive monocyte-like cells were more frequent in the tumour-infiltrating fraction (17%) than ascites (7%) or blood (12%). B lymphocytes were infrequent in solid tumours and difficult to assess in ascites. Histiocyte-like macrophages were present in the higher-velocity tumour-cell containing fractions of both solid and ascitic material. The variation in infiltrating cells between patients and between tumour and ascites of the same individual was marked.     

Correlation of tumor necrosis factor and prostaglandin E2 production of monocytes in bladder cancer patients

Patients with advanced malignant neoplasms have a variety of abnormal monocyte and lymphocyte functions. The authors examined tumor necrosis factor (TNF) and prostaglandin E2 (PGE2) production of monocytes in 48 patients with bladder cancer and 16 control subjects. Monocytes were isolated from peripheral blood mononuclear cells by adherence to plastic tissue. They were cultured with lipopolysaccharide for 24 hours, and the culture supernatant was obtained. The TNF was measured by enzyme immunoassay using anti-recombinant human TNF antibody, and PGE2 was measured by radioimmunoassay. As a result, in high-stage bladder cancer patients, there was a significant inverse correlation between TNF and PGE2 production of monocytes. However, there was no significant correlation in control subjects and low stage patients. Accordingly, some patients with high-stage bladder cancer had higher TNF production but lower PGE2 production of monocytes, and vice versa.     

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia

Bone marrow and peripheral blood samples from 362 patients with acute lymphoblastic leukaemia (ALL) proliferating cell and 90 patients with acute myeloid leukaemia (AML) were analysed for S-phase fractions, Ki67 antigen, and proliferating cell nuclear antigen expression. The S-phase fractions were correlated with in vitro drug resistance to 15 different anticancer agents. Leukaemia cells isolated from bone marrow had higher S-phase fractions than leukaemia cells isolated from peripheral blood (in initial ALL, median values resp. 6.9 and 2.7%, in initial AML resp. 5.3 and 1.3%; both P<0.01). Relapse ALL samples derived from bone marrow showed increased S-phase fractions (median 9.9%) compared with initial ALL samples (median 6.9%; P<0.01). ALL samples obtained at initial diagnosis showed higher S-phase fractions (median 6.9%) and higher Ki67 expression (median 30%) than initial AML samples (median resp. 5.3 and 14%; both P<0.05). The S-phase fractions were not related to white blood cell count, age, or gender. Within initial ALL, the S-phase fraction correlated significantly but modestly strong (rho=0.3-0.5; P<0.05) with sensitivity to antimetabolites (cytarabine, mercaptopurine, thioguanine), L-asparaginase, teniposide, and vincristine. Similar results were found within subgroups of initial ALL (nonhyperdiploid and common/precursor-B-lineage ALL). In relapsed ALL and AML such correlations were not found. In conclusion, cell proliferation differs between leukaemia subgroups and increased proliferation is associated with increased in vitro sensitivity to several anticancer agents in initial ALL.     

Enhanced production of interleukin 6 in peripheral blood monocytes stimulated with mucins secreted into the bloodstream.

PURPOSE: It has been reported that tumor progression is correlated with the serum level of interleukin 6 (IL-6). The purpose of this study was to investigate by what mechanism, other than production from tumor cell, the serum level of IL-6 is elevated in the tumor-bearing state. EXPERIMENTAL DESIGN: Monocytes from healthy donors were cultured in the presence of sera from colon cancer patients, and the activity to elevate IL-6 production was estimated. This activity of serum was also examined after various biochemical treatments. RESULTS: When monocytes from healthy donors were cultured in the presence of sera from patients with colon cancer, secretion of IL-6 from the cells was markedly elevated. Serum proteins were fractionated on Sepharose 4B and the activity to elevate IL-6 production was found in the excluded fractions. Sialyl Tn antigen was detected in these same fractions. By excluding some mucins from the serum, the inducing activity was reduced to 40% of the original level. Furthermore, we purified mucins from the conditioned medium of colon cancer cells. Production of IL-6 was effectively elevated by a small amount of purified mucins in a dose-dependent manner. When the inducing activity was examined in the presence of binding or competitive inhibitors to the scavenger receptor, the effect was remarkably reduced. CONCLUSIONS: Mucins secreted from colon cancer cells into the bloodstream induce production of IL-6 in peripheral blood monocytes through the scavenger receptor, which may be responsible for the high level of serum IL-6 in colon cancer patients.     

肿瘤转移患者外周血细胞的某些生物学特征

目的:探讨肿瘤转移患者外周血细胞的生物学变化特征。方法:用流式细胞术对396例肿瘤转移患者和371例非转移患者外周血细胞的DNA倍体、凋亡水平和增殖活性进行了对比分析。结果:在有肿瘤转移的396例患者外周血细胞中,有45例出现了DNA异倍体,其检出率为11.36%;DNA异倍体患者血细胞的DNA指数(DNAindexDI)值为1.25±0.26。另371例非转移患者未发现DNA异倍体细胞。肿瘤转移者的细胞凋亡率和S期细胞比率均显著高于非转移者P<0.05,多器官转移者的细胞凋亡率和S期细胞比率也显著高于单器官转移者P<0.05。结论:肿瘤转移能引起患者外周血细胞中出现DNA异倍体,并使血细胞凋亡水平和增殖活性上调。     

Renal cell carcinoma induces interleukin 10 and prostaglandin E2 production by monocytes.

Immunotherapy with interleukin 2 (IL-2) is not an effective anti-cancer treatment in the majority of patients with renal cell carcinoma (RCC), suggesting that the activation of cytotoxic T cells or NK cells may be impaired in vivo in these patients. The production of immunosuppressive factors by RCC was investigated. Using immunohistochemistry, IL-10 was detectable in 10 of 21 tumour samples tested. IL-10 was undetectable in the supernatant of cell lines derived from these RCCs. However, these cell lines or their conditioned medium (RCC CM), but not normal renal epithelial cells adjacent to the RCC or breast carcinoma cell lines, were found to induce IL-10, as well as prostaglandin E2 (PGE2) and tumour necrosis factor (TNF)alpha production by autologous or allogeneic peripheral blood mononuclear cells (PBMCs) and monocytes. IL-10 production induced by RCC CM was found to be dependent on TNF-alpha and PGE2 since an anti-TNF-alpha antibody (Ab) inhibited 40-70% of IL-10 production by monocytes, and the combination of anti-TNF-alpha Ab and indomethacin, an inhibitor of PGE2 production, inhibited 80-94% of RCC CM-induced IL-10 production by monocytes. The RCC CM of the five cell lines tested were found to induce a down-regulation of the expression of HLA-DR and CD86, as well as a strong inhibition of mannose receptor-dependent endocytosis by monocytes. The blockade of HLA-DR and CD86 expression was partially abrogated by indomethacin and anti-IL-10 Ab respectively, and completely abrogated by an anti-TNF-alpha Ab. The inhibition of mannose receptor-dependent endocytosis was partially abrogated by an anti-IL-10 Ab and completely abrogated by an anti-TNF-alpha Ab. These results indicate that RCCs induce IL-10, PGE2 and TNF-alpha production by monocytes, which down-regulate the expression of cell-surface molecules involved in antigen presentation as well as their endocytic capacity.     

Establishment, characterization and differentiation induction of a new human diploid myelomonocytic cell line (HL-92) derived from a patient with acute myelomonocytic leukemia

With very few exceptions, it has not been possible to grow human myeloid cells for long periods in culture. We have recently developed techniques enabling the long-term in vitro propagation of normal immature myeloid cells from fresh foetal cord blood and monocytes from normal adult peripheral blood, and have utilized these procedures to initiate cultures of fresh peripheral blood leukocytes from leukemic donors. In four of 26 leukemic samples tested, leukocyte replication beyond that obtained in control cultures was observed, and in one of these HL-92, derived from the peripheral blood of a patient with acute myelomonocytic leukemia, the culture has continued to replicate slowly for over 2 years under the special growth conditions. Morphological, cytochemical, immunological and functional studies show that the culture consists predominantly of immature myeloid cells (myeloblasts through to myelocytes) but also contains some mature neutrophils and monocytes. At least a portion of HL-92 cells express Fc and complement receptors, contain histocompatability locus antigens, including HLA-DR, and release GM-CSA, low levels of PGE and lysozyme. HL-92 cells can be induced with DMSO or RA to differentiate into mature neutrophils (an increase from 20 to 70% of the cell population) as determined by morphology, by an increase in phagocytic cells, and superoxide anion production. Fresh leukocytes from the patient's bone marrow appeared to have a diploid karyotype. However a consistent chromosomal abnormality observed in HL-92 was a deletion in the long arm of chromosome 11 [del(11)(q23)]. This is consistent with recent observations in monocytic leukemia. Since the few other established human myeloid cell lines have various chromosomal abnormalities, and some respond to differentiation inducers, while others do not, there appears to be no detectable common chromosome change required either for in vitro growth of myeloid cells or their response to inducers of differentiation. These cell lines and the application of the techniques described here for the growth of myeloid cells from other leukemic or normal sources should be helpful in the study of normal and leukemic myeloid cell growth and differentiation.     

Acetylcholinesterase (AchE) activity of lymphocytes in chronic lymphoid leukemia (CLL)

The specific AchE (EC 3.1.1.7) activity of lymphocytes from the peripheral blood of normal donors was determined. On Leukopak filter the isolated T lymphocytes showed activity, whereas in stepwise Percoll gradient, population T_LD displayed enzyme activity. The T_HD and B cells were inactive. [Szelényi J. G., Bartha E. & Hollán S. R. (1982) Br. J. Haemat. 50, 241]. A mixed cell population derived from CLL patients had significantly lower enzyme activity than normal. With the progress of B-cell proliferation AchE activity decreased in parallel with the number of T cells. The sp. act. of T_LD population isolated from CLL patients was the same as that of normal donors whereas their B cells were inactive.     

Determination of DNA synthesis, estrogen receptors, and carcinoembryonic antigen in isolated cellular subpopulations of human breast cancer

Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.     

Superoxide anion-generating activity of polymorphonuclear leukocytes and monocytes in patients with lung cancer.

Superoxide anion (O2-) production by polymorphonuclear leukocytes (PMN) and monocytes (MN) was measured in the peripheral blood of 70 patients with lung cancer. The O2- production by these cells was decreased in many, but not all, patients. The incidence of patients with lower O2- production increased as the stage advanced. The correlation between O2- production by these cells and peripheral blood smears was evaluated in patients with cancer. Patients with 80% granulocytes and 40% monocytes or more in their peripheral blood had a significantly lower O2- production by PMN and MN compared with those with less than 80% granulocytes and 40% monocytes, respectively. These results indicate that an abnormally increased number of granulocytes and monocytes in the peripheral blood of patients with cancer may depress immunoregulatory function. In addition, decreased O2- production by these cells should be considered when assessing the defense mechanisms and susceptibility to infection of these patients.     

Cytotoxicity of human rosette-forming blood lymphocytes on cultivated human tumor cells

Human peripheral blood lymphocytes from seven cancer patients were separated into two fractions, one rich in lymphocytes which could spontaneously form rosettes with sheep erythrocytes (T-cells) and another poor in rosette-forming cells. These two fractions were then tested for cytotoxic activity on cultivated tumor cells of the same histological type as the tumors of the respective lymphocyte donors. It was shown that T-cell rich fractions had a higher cytotoxic activity than did the T-cell poor fractions from the same patients. The findings indicate that human T-cells can be cytotoxic, in vitro, against cultivated cells carrying tumor-associated antigens.     

Low molecular thymic peptides improve the deficient immunocytotoxicity of mononuclear cells from tumor patients in vitro

We studied, in vitro, the stimulating effects of a commercial preparation of low molecular thymic peptides (TP) on the immunocytotoxicity of peripheral blood lymphocytes and monocytes from patients with breast tumors, melanoma and colorectal tumors. On average, tumor patients showed a lower natural killer (NK), lymphokine (IL-2) activated killer (LAK) cell and basic tumoristatic activity of monocytes, compared with healthy donors. There was no correlation between the NK-cell number and the NK-cell activity of the tumor patients. The TP showed no effects on the NK-cell activity in any group, yet elevated the deficient LAK-cell activity of tumor patients and that of healthy donors. On monocytes, TP enhanced the deranged tumoristatic activity only in tumor patients, while being slightly inhibitory on control monocytes. Dividing the donors on the basis of the TP effects on cytotoxicity of the mononuclear cells into TP-nonresponders and TP-responders, a higher number of TP-responders was found among tumor patients, compared with healthy donors. Moreover, a higher number of TP-nonresponders were observed with lymphocytes from colorectal tumor patients at advanced tumor stage. Therefore, on the basis of the applied immunocytotoxic assays, these results may provide a basis for selecting tumor patients, who may respond to TP in immunotherapy protocols.     

Deficient production of tumor necrosis factor by peripheral-blood monocytes in chronic lymphocytic leukemia

The production of tumor necrosis factor (TNF) by lipopolysaccharide (LPS)-triggered peripheral-blood mononuclear cells (PBM) was investigated in 23 patients with untreated B-cell chronic lymphocytic leukemia (B-CLL) and 14 control donors. Cells were stimulated at concentrations that reflect cell density in peripheral blood. Under these conditions, PBM from 11/23 of the CLL patients produced at least 10-fold less TNF as compared with controls. Monocyte numbers were decreased in percentage, while absolute numbers (normal range 233 +/- 120 X 10(3)/mm3) were decreased only in 2, normal in 17 and increased in 4 patients indicating that the deficiency is not a result of monocytopenia in most patients. Cell separation experiments indicate that after removal of leukemic B cells, percentages of monocytes return to control range and TNF production is improved (7/7). In mixing experiments, we found a suppression of TNF production in control mononuclear cells by CLL cell samples (75 X 10(6) cells/ml) in 5/19 cases, while control cells from thymus exhibited no or little suppression in these conditions. In 2-chamber experiments, leukemic samples suppress TNF production by normal monocytes across a 0.45 micron membrane indicating that a soluble factor is responsible for suppression. The factor exhibits higher stability in serum-free conditions and its molecular weight is below 20 kDa. Prostaglandins are not involved, since indomethacin did not abrogate suppression.