Gamma-linolenic acid induces apoptosis in B-chronic lymphocytic leukaemia cells in vitro.

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5 microg-60 microg) was: 42%-95%. In the presence of GLA 5 microg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15 microg/ml: P=0.045; 0.027 and 0.022, respectively. At 30 microg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30 microg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30 microg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20 microg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B- and T-cells cells in vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B- and T-cells.     

The levels of TNF alpha, IL4 and IL10 production by T-cells in B-cell chronic lymphocytic leukaemia (B-CLL)

Tumour necrosis factor alpha (TNF alpha), interleukin (IL) 4 and IL10 are important for the growth and survival of the leukaemic cells in B-cell chronic lymphocytic leukaemia (B-CLL). The present study investigates the production of TNF alpha, IL4 and IL10 in patients with B-CLL. Significant increases in the TNF alpha and IL4 mean levels compared to normal control CD2(+)-cells were observed for B-CLL lymphocytes (TNF alpha: P=0.0004 and IL4: P=0.0026). By contrast, the mean level of IL10 produced by purified B-CLL CD2(+)-cells was significantly lower than that seen with normal control T-cells (P=0.0136). No significant difference in the percentage (%) of T-cells that expressed cytoplasmic TNF alpha, IL4 and IL10 was observed between B-CLL and normal T-cells. However, a significant increase in the mean level of intracellular TNF alpha and IL4 expression was observed in B-CLL compared with normal control T-cells (TNF alpha: P=0.031; IL4: P=0.0027). The increased expression of cytoplasmic TNF alpha and IL4 appeared to be associated with increased cytokine production in the tested samples. The differences observed with some B-CLL cases in the production of TNF alpha, IL4 and IL10 by peripheral blood T-cells may suggest survival mechanisms for the leukaemic cells in these patients.     

γ-Linolenic Acid Induces Apoptosis in B-Chronic Lymphocytic Leukaemia Cells in Vitro

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5μg-60μg) was: 42%-95%. In the presence of GLA 5μg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15μg/ml: P=0.045; 0.027 and 0.022, respectively. At 30μg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30μg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30μg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20μg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B-and T-cells cells in-vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B-and T-cells.     

Reactivity of the Endothelial Cell Marker EN4 With Malignant B-Cells in Low-Grade Malignant Lymphomas and with Subpopulations of B- and T-Cells in Blood and Reactive Lymphoid Tissue

Expression of the endothelial cell marker EN4 was studied by immunohistochemical staining in 56 biopsies of malignant Non-Hodgkin lymphomas (NHL), Hodgkin's disease, reactive lymphatic tissue and in non-lymphatic organs and by flow-cytofluorometry in blood and tonsil cell suspensions.

In addition to positivity in endothelial cells, MAb EN4 reacted with numerous leukocytes localized in perivascular zones, mostly T lymphocytes and monocytes/macrophages. Neoplastic B cells of low-grade lymphocytic and centrocytic NHL, immunocytomas and neoplastic T (CD3+/CD4+) cells from one lymphoblastic lymphoma were also positive. In contrast, however, centroblastic/centrocytic NHL, high grade malignant B-cell lymphomas and Reed-Sternberg cells in Hodgkin's disease were negative.

The majority of B-cells and CD8 positive T-cells both in blood and tonsil cell suspensions were EN4 positive. CD4+ cells were EN4 positive but to a lesser extent. All CD14 positive monocytes, all granulocytes and the majority of CD56 positive cells (NK cells, cytotoxic T-lymphocytes) expressed EN4.

These observations suggest that EN4 epitope could be involved in the mechanisms of leukocyte traffic and their adhesion to endothelium.     

Bcl-2 and bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients

Chlorambucil and other cytotoxic drugs kill cells, non-selectively, by inducing apoptosis. In this study, we measured the apoptotic response to chlorambucil in T- and B-cells from untreated B-CLL patients and T-cells, from normal control subjects. We found increased chemosensitivity in the T-cells of B-CLL patients compared to the controls (P=0.0002). The chlorambucil ID(50) values for T-cells from B-CLL patients showed a direct correlation with Bcl-2 expression (P=0.002) and an inverse correlation with CD3 cell count (P<0.0001), suggesting a trend of increasing chemosensitivity and decreasing Bcl-2 expression with an elevated T-cell count. There was no differential expression of Bcl-2 or Bax between the CD4(+) and CD8(+) cells of B-CLL patients, isolated by immunomagnetic separation. We found correlations in the leukaemic B-cells between chlorambucil ID(50) values and both Bcl-2 expression (P=0.006), and Bcl-2/Bax ratios (P=0.002), suggesting a role for the Bcl-2/Bax ratio in predicting the response of untreated CLL patients to cytotoxic treatment. Chlorambucil produced almost identical changes in Bcl-2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between Bcl-2 and chemosensitivity confirms a pivotal role for Bcl-2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage.     

Exogenous interleukin 2 (IL 2) reconstitutes deficient in vitro T-cell growth of patients with chronic lymphocytic leukemia of B-cell lineage (B-CLL)

Mechanisms of tumor growth are concerned with the possibility that neoplasms are disturbed in their response to growth factors. Interleukin 2 (IL 2) is secreted by activated T-cells and is required for autocrine T-cell growth. Controversy exists as to the functional capacity of T-lymphocytes in patients with B-cell chronic lymphocytic leukemia (CLL). We studied 14 patients with previously untreated, early CLL. Data were compared to 16 healthy individuals. Immunophenotyping showed that T-cells in B-CLL are highly activated as evidenced by HLA-DR and IL 2 receptor expression. Membrane receptors for IL 2 on the patients' T-cells were overexpressed. The T4:T8 ratio was 1.5. T-cells for in vitro studies were isolated by their ability to form E-rosettes with AET and repeated Ficoll density gradient centrifugation. Purified T-cells were grown on semisolid agar and their capability to form colonies investigated using a variety of exogenous stimulants. It was found that patients with CLL showed a pronounced impairment in forming T-cell colonies. However, colony formation was restored to normal by adding exogenous recombinant IL 2, indicating that endogenous IL 2 production of T-cells is deficient. This finding might have implications for the therapeutical use of IL 2 in CLL patients.     

Lymphocyte response to pokeweed mitogen in chronic lymphocytic leukemia

The response of lymphocyte subpopulations to pokeweed mitogen (PWM) was studied in normal volunteers and patients with B-cell chronic lymphocytic leukemia (CLL). Since unfractionated peripheral blood mononuclear (PBM) cells from CLL patients consist of a markedly increased proportion of B-lymphocytes and a decreased proportion of T-lymphocytes, enriched fractions of CLL B-cells and CLL T-cells were cultured in 1:1 proportions in autologous and allogeneic combinations with normal B-cell and T-cell-enriched fractions. Cultures containing normal B-cells with either autologous or allogeneic normal T-cells responded well to PWM. CLL T-cells were capable of providing a helper function for both proliferation and differentiation of normal B-cells, which was not significantly different from that provided by allogeneic normal T-cells. CLL B-lymphocytes were unresponsive to PWM when cultured in the presence of either autologous CLL T-lymphocytes or allogeneic normal T-lymphocytes. The responsiveness of CLL B-cells was not restored by the addition of normal peripheral blood monocytes to the cultures. These experiments indicate that there is an intrinsic B-cell defect which prevents CLL B-lymphocytes from responding to PWM.     

Differential expression of TRAP Isoenzyme in B-CLL Cells Treated with Different Inducers

Purified B-cells from normal tonsils and from the peripheral blood of eight patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with the protein kinase C (PKC) activators TPA, Bryostatin 1 (Bryo), mezerein, with the calcium ionophore A23187, and with the cytokines interleukin-1/2, interferon-alpha/gamma, tumor necrosis factor. The induction of the lysosomal enzyme tartrate-resistant acid phosphatase (TRAP) was examined at the RNA (by Northern blotting analysis) and the protein level (by isoelectric focusing). TRAP mRNA and protein were induced by treatment with PKC activators while the combination of PKC stimulator and calcium ionophore was not effective, TRAP mRNA was detected as early as 2 hours after initiation of the culture. This induction of positivity for TRAP which is the enzymatic hallmark of hairy cell leukemia (HCL) by TPA or Bryo is consistent with the previously reported acquisition of HCL-type cellular features in TPA-driven CLL cells; CLL cells exposed to the double stimulus of TPA or Bryo + A23187 have previously been described to differentiate to plasmacytoid cells which is in accord with their remaining TRAP negative. The present data provide evidence that 1) B-CLL cells can be induced by direct stimulation of PKC to convert to HCL-type cells; 2) TPA/Bryo-exposed B-CLL cells display phenotypes different from those of cells treated with combinations containing calcium ionophore; 3) the phosphoinositol signal transduction pathway distal to PKC can be activated effectively in B-CLL cells; 4) Bryo, a new natural PKC activator, induces responses in CLL similar to those caused by TPA; 5) TRAP is an inducible indicator of cellular activation. These observations provide support for the idea that HCL and HCL-like cells might differentiate along a separate branch of B-cell lineage and might represent mature “activation end stage” cells.     

Kinetics of protein a activation of mononuclear cells from patients with chronic lymphocytic leukemia — I. CLL B-cells are not intrinsically unresponsive to staphylococcal protein A

The response of lymphocyte subpopulations to staphylococcal Protein A (SPA) was studied in patients with B-cell chronic lymphocytic leukemia (CLL) and normal volunteers. The kinetics of the proliferative response, optimal dose and sera requirements were determined. Of 92 experiments conducted in 60 patients, SPA induced peripheral blood mononuclear cells (PBMC) proliferation in 81.5% of cases studied. The mean proliferative response of CLL PBMC was significantly lower than normal PBMC, 5823 vs. 30,364 cpm. However, enriched CLL B-cells failed to respond to SPA compared to normal enriched B-cells, with mean cpm 444 vs. 6438 respectively. As PBMC from CLL consist of increased proportions of B-cells and decreased proportions of T-cells, enriched CLL B-cells were cultured at 1 : 1 ratio with autologous or allogeneic normal T-cell enriched fractions. CLL B-lymphocytes were stimulated by SPA when cultured in the presence of T-lymphocytes, indicating a T-helper defect in the B-CLL proliferative response to SPA, rather than an intrinsic inability of CLL B-cells to proliferate. These observations are of import for further studies of CLL B-lymphocyte function, cytogenetics, and establishment of CLL B-cell lines in culture.     

Immunoreactive lactoferrin in resting, activated, and neoplastic lymphocytes

Lactoferrin (Lf) in lymphocytes was assessed with immunofluorescence/flow cytometric technique. Surface Lf was detected primarily among B-cell-enriched preparations. Tonsillar B-cells of different densities expressed surface Lf similarly. Very small percentages of CALLA+ ALL, HCL, or EBV-transformed B-cells expressed surface Lf, whereas B-CLL lymphocytes had the highest percentages of surface Lf positivity. Few resting, cultured, or neoplastic T-lymphocytes expressed Lf. The pattern of immunofluorescence and analyses of surface and total cellular immunoreactive Lf indicated that Lf is associated primarily with the lymphocyte surface. The percentage and/or intensity of surface Lf-specific fluorescence were not significantly altered in B- or T-cells by incubation with physiologic concentrations of differric Lf, and the percentages of Lf-positive cells detected in respective subjects remained stable over time. Surface Lf positivity was unrelated to the expression of other surface antigens (except those marking B- or T-cell lineage) or cell cycle. Expression and/or binding of Lf in B-lymphocytes may become increased during certain stages of cell maturation.     

CD38 Expression Correlates with Adverse Biological Features and Predicts Poor Clinical Outcome in B-Cell Chronic Lymphocytic Leukemia

CD38 identifies a surface molecule with multi-functional activity. Its prognostic importance in B-cell chronic lymphocytic leukemia (B-CLL) is currently under investigation in view of the fact that two different groups have recently indicated that CD38 expression could be an independent prognostic marker in B-CLL.

We analyzed the clinico-biological features of 61 immunologically typical (CD5+CD23+) B-CLL patients stratified according to the CD38 expression. Twenty-two (36%) patients expressed CD38 in more than 30% of CD19-positive cells and were considered as CD38-positive B-CLL. Atypical morphology (p 0.02), peripheral blood lymphocytosis (p 0.01) and diffuse histopathologic bone marrow pattern (p 0.003) were findings found to be closely associated with CD38 expression. On the other hand, A and B Binet stages (p 0.02) and interstitial bone marrow involvement (p 0.005) were more represented in the CD38-negative B-CLL group. Trisomy 12 was detected more frequently in the CD38-positive B-CLL group, while 13q14 deletions mainly occurred in CD38-negative group (p 0.005). Finally, median survival of CD38-positive B-CLL patients was 90 months, while it was not reached at 180 months in CD38-negative patients.

Taken together, our data strongly suggest that the evaluation of CD38 expression may identify two groups patients with B-CLL greatly differing in their clinico-biological features.     

Fc receptor-like 1-5 molecules are similarly expressed in progressive and indolent clinical subtypes of B-cell chronic lymphocytic leukemia

Fc receptor-like (FCRL) 1-5 molecules are exclusively expressed in B-cells and have recently been considered as potential targets for immunotherapy of B-cell malignancies. In this study, the expression pattern of FCRL1-5 molecules was investigated in Iranian patients with B-cell chronic lymphocytic leukemia (B-CLL). Our RT-PCR results have demonstrated that all FCRL molecules, except FCRL4, were expressed in the vast majority of the patients with B-CLL. However, comparison of the relative mRNA expression levels of FCRL between B-CLL (n = 86) and elderly normal subjects (n = 10) revealed significantly lower expression levels of FCRL1 (p < 0.0001), FCRL3 (p = 0.01) and FCRL4 (p = 0.002), but not FCRL2 or FCRL5, in cases with B-CLL. No significant differences were observed between the indolent and progressive subtypes of patients with B-CLL. Comparison between the mutated and unmutated subtypes revealed a significantly higher expression level of FCRL3 (p = 0.017) in patients with mutated CLL. Surface and intracytoplasmic expression of FCRL1, 2, 4 and 5 in leukemic cells of 12 patients by flow cytometry revealed similar results to those obtained by RT-PCR with a few exceptions. Thus, while FCRL4 was expressed in only 2 samples at intracytoplasmic level, FCRL1 and 2 were expressed in the majority of samples, both at surface and intracytoplasm. FCRL5 protein was also detected in 10 samples, but surface expression was confirmed in only 2. Analysis of B-cells from 5 normal subjects by flow cytometry revealed higher expression levels of FCRL molecules compared to CLL. Our results indicate differential expression of FCRL molecules in B-CLL and suggest the potential implication of FCRL1 and 2 for immunotherapeutic interventions.     

Morphologically Typical and Atypical B-Cell Chronic Lymphocytic Leukemias Display a Different Pattern of Surface Antigenic Density

Recent evidences suggest that B-cell chronic lymphocytic leukemia (B-CLL) may have heterogeneous biological and clinical features. Immunological phenotype may be useful for distinguishing these different forms of disease.

We used a quantitative flow cytometric approach to analyze the expression of several membrane molecules (CD19, CD20, CD22, CD23, CD11c, CD5, CD79b) commonly used to diagnose and characterize B-CLL in a choort of 84 consecutive B-CLL patients diagnosed according to morphological and immunological findings. We found that morphologically so-called “atypical” B-CLL displayed a significantly higher number of CD20 and CD22 molecules than typical forms. On the other hand, CD19 was found to be more expressed in typical B-CLL, although without reaching statistical significance. Finally, no difference was detected with respect to CD23, CD79b, CD11c and CD5 number of molecules/per cell between typical and atypical B-CLL. Other clinico-biological features, such as surface membrane immunoglobulin density, percentage of CD79b and FMC7 expression, peripheral blood lymphocytosis, trisomy 12 and advanced clinical stages were also found to be more frequent in atypical B-CLL. In conclusion, our data confirm the hypothesis that atypical B-CLL is a disease sustained by more mature B-cells, closely related but, at the same time, clearly distincted from neoplastic cells of typical B-CLL.     

Morphologic and cytochemical comparison of human lymphoblastoid T-cell and B-cell lines: light and electron microscopy.

Human lymphoblastoid cell lines characterized as T- or B-cells by various markers were compared morphologically and cytochemically by light and electron microscopy. Distinct differences in nuclear morphology, amount of cytoplasm, pyroninophilia, and periodic acid-Schiff (PAS) staining enabled us to discriminate between T- and B-cell lines. T-cells had nuclei with an irregular configuration, stippled heterochromatin, and small or absent nucleoli. The scanty cytoplasm of T-cells contained intensely stained, PAS-positive globules and was less pyroninophilic than the cytoplasm of B-cells. B-cells had more rounded, uniform, vesicular nuclei with prominent nucleoli and peripheral heterochromatin. The cytoplasm of B-cells was abundant and strongly pyroninophilic. Transmission electron microscopy generally confirmed these morphologic differences. These findings supported our contention that consistent cytologic features concordant with immunologic markers make it possible to identify certain lymphomas as being of B- or T-cell origin on purely morphologic grounds.     

Activation of mouse macrophages by alkylglycerols, inflammation products of cancerous tissues

Alkylglycerols, inflammation products of lipids in cancerous tissues, are potent macrophage stimulating agents. Administration of small amounts (10-100 ng) of alkylglycerols to mice greatly enhanced macrophage activation for Fc-mediated ingestion activity at the 5th day posttreatment. Dose effect analysis revealed that dodecylglycerol (DDG), one of the alkylglycerols, stimulates macrophages most effectively at the dose of 100 ng/mouse. Administration of lower concentrations of a longer carbon chained alkylglycerol, sn-3-octadecylglycerol (batyl alcohol), to mice produced a similar activation of macrophages. In vitro incubation of mouse peritoneal cells with 50 ng DDG/ml efficiently stimulated macrophages for Fc-mediated ingestion activity. However, in vitro treatment of macrophages alone with DDG was unable to stimulate ingestion activity. When a mixture of macrophages and nonadherent (B and T) cells was treated with DDG, a greatly enhanced Fc-mediated ingestion was observed at about 3 h posttreatment, suggesting that nonadherent cells contributed to the activation of macrophages. Since coincubation of these cells with DDG is required for macrophage activation, stepwise stimulation processes by exchanging signaling factor(s) among these cell types were considered for the developmental mechanism of ingestion capacity of macrophages. When a conditioned medium of DDG-treated B- or T-cells was admixed with macrophages and incubated for 3 h, no significantly enhanced ingestion activity of macrophages was observed. Thus, exchange of signaling factor(s) among B- and T-cells was analyzed by transferring conditioned media of DDG-treated B- or T-cells to untreated T- or B-cells. When the resultant (treated B-cells----untreated T-cells conditioned medium was admixed with untreated macrophages and incubated for 3 h, a markedly enhanced Fc-mediated ingestion was observed. However, no significant increase in ingestion activity was found in macrophages incubated with the treated T-cell----untreated B-cell conditioned medium. Therefore, we concluded that DDG-treated B-cells initiated macrophage activation processes by releasing and transmitting a signaling factor(s) to T-cells, and in turn the T-cells modified the factor or produced a new factor(s) capable of the ultimate stimulation of macrophages for ingestion capability.     

In vitro beta 2-microglobulin (beta 2m) secretion by normal and leukaemic B-cells: effects of recombinant cytokines and evidence for a differential response to the combined stimulus of phorbol ester and calcium ionophore

Due to the increasing therapeutic use of immunoregulatory agents and the potential effects on cellular function, we examined the modulation of in vitro beta 2-microglobulin (beta 2m) production rates by 'normal' tonsil and leukaemic B-cells in response to a number of these agents. Tonsil B-cells responded to phorbol ester (TPA) by an increased beta 2m production rate, which was further enhanced by the combined stimuli of TPA plus the calcium ionophore A23187. In marked contrast, however, lymphocytes from a majority (8/11) of B-cell malignancies showed a suppression of the TPA-induced beta 2m production rate in response to the combined TPA/A23187 stimulus. These different responses of 'normal' and malignant B-cells were not apparent when IgM production rates were examined. The recombinant cytokines IL-1, IL-2, IFN-alpha, IFN-gamma and TNF also enhanced beta 2m production rates of both normal and leukaemic B-cells, but to a considerably lesser extent than did TPA. Bryostatin-1 increased beta 2m production to a level intermediate between that obtained by TPA and the cytokines. It is suggested that beta 2m production rates may correspond to the degree of B-cell differentiation, and/or to the degree of cellular 'activation'. The results further indicate that the in vitro measurement of beta 2m production provides a different index of the cellular response than that obtained by the conventional measurement of IgM production.