Interleukin 4 production by peripheral blood lymphocytes in patients with classical Hodgkin lymphoma.

Although the production of interleukin (IL) 2 and interferon (IFN) gamma by peripheral blood lymphocytes in patients with Hodgkin lymphoma (HL) is well documented, the synthesis of IL4 has not been investigated before. The present study examines the production of IL4 by 2-day phytohaemaglutinin (PHA)-stimulated peripheral blood (PB) cells in HL and correlates the cytokine levels with the proportion of the different T-cell sub-populations. We observed a significant increase in the mean level of production of IL4 in patients with HL when compared with normal controls. The increased amount of IL4 in patients with HL correlated significantly with the proportion of the CD3(+)CD8(+) cells but not with CD3(+)CD4(+). The intensity of cytoplasmic IL4 (expressed as relative median fluorescence (RMF)) was significantly higher in the CD3(+)CD8(+) cells of the patients with HL compared with the CD3(+)CD4(+) sub-population, or with the normal CD3(+)CD8(+) cells and correlated with the levels of IL4 release in culture supernatants. In conclusion, there is increased production of IL4 by PHA-activated PB lymphocytes in HL. The CD3(+)CD8(+) T-cell population appears to be responsible for this increased synthesis.

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.

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

AIMS: To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS: The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS: Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS: B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis.

Virulence of Listeria spp.: course of infection in resistant and susceptible mice.

The virulence of representative strains of the five species of Listeria monocytogenes sensu lato was compared in C57BL/6 and BALB/c mice in terms of LD50 values and of bacterial growth kinetics and histological changes in mouse livers. L. monocytogenes sensu stricto and L. ivanovii showed relatively low LD50 values and much bacterial growth for 2-3 days before viable counts declined. Histological changes in L. ivanovii infection resembled those caused by L. monocytogenes, with early development of neutrophil-rich micro-abscesses and hepatocyte necrosis followed by macrophage infiltration and formation of granulomas. By contrast, L. innocua, L. welshimeri and L. seeligeri were almost entirely avirulent as shown by high LD50 values, early elimination of viable bacteria and no evidence of growth. Histological changes consisted only of slight, transient infiltration of the liver with neutrophils. Both groups of bacteria were seen infrequently in Kupffer cells early in infection, but only the highly virulent species appeared to replicate. LD50 values for L. monocytogenes and L. ivanovii were (10-20)-fold greater, and for the less virulent bacteria at least two-fold greater, in C57BL/6 than in BALB/c mice. This difference in host susceptibility was not reflected in detectable histological differences between the two mouse strains.

Modulation of apoptosis with cytokines in B-cell chronic lymphocytic leukaemia.

In B chronic lymphocytic leukaemia (B-CLL) non-proliferating peripheral blood (PB) B cells have a long life span in vivo. In cultures, these cells die spontaneously by apoptosis. Interleukin (IL) 4 inhibits spontaneous apoptosis (SA) and promotes survival of B-CLL B cells in vitro. No such effect is observed in PB B cells from normal healthy donors. The anti-apoptotic effect of IL4 is independent of mitogen-induced cell activation but depends on the concentration of IL4. The protective effect of IL4 is specific and it is significantly reduced or abolished with anti-IL4 antibody. Interferon (IFN)-gamma and alpha- IFN also protect B-CLL B cells from apoptosis in vitro. Sera from B-CLL patients have increased levels of IFN-gamma when compared with sera from healthy donors. In addition, B-cells in B-CLL express detectable levels of IFN-gamma mRNA. Other cytokines, namely ILl, IL2, IL6 and IL7 do not affect SA of B-CLL B cells. By contrast, IL5 and antibody to apolipoprotein-1 (APO- 1) receptor increase SA significantly and in a dose-dependent manner. Interleukin 4 protects B-CLL B cells from IL5-, anti(alpha) APO-1- and steroid-induced apoptosis. The mode of action of the cytokines inducing apoptosis or protecting B-CLL B cells from dying is largely unknown. Recently the bcl-2 proto-oncogene has been associated with prolonged cell survival. However, the involvement of bel-2 in spontaneous, cytokine-induced or steroid-induced apoptosis in B-CLL has been controversial. Some authors have reported down-regulation of bcl-2 protein expression in B-CLL B-cells undergoing SA or in steroid-treated cells with IL4 preventing this down-regulation. By contrast, others observed no significant loss of bcl-2 protein expression in steroid-, alpha-APO-1 - and IL5-treated cells when compared with untreated or fresh cells. Also, no correlation between bcl-2 protein expression and protection with IL4 has been reported. In conclusion, in B-CLL IL4, IFN-gamma and alpha-IFN promote the survival of the leukaemic cells. These cytokines may therefore be involved in the pathogenesis of the B-CLL.

Expression and production of interleukin 4 in B-cell chronic lymphocytic leukaemia.

Interleukin (IL) 4 is a T-cell derived pleiotropic cytokine whose properties include alterations of B-cell function. In B-cell chronic lymphocytic leukaemia (B-CLL), IL4 is involved in the mechanism of survival of the leukaemic B-cells. The present study examines the expression and production of IL4 by B- and T-lymphocytes derived from patients with B-CLL and provides evidence that IL4 is not an autocrine factor in B-CLL. Freshly isolated B-CLL cells enriched for B- and T-cells did not express mRNA for IL4 but expressed mRNA for IL4 receptor (IL4R). Activation of B-cells with phorbol ester and calcium ionophore and of T-cells with phytohaemaglutinin (PHA) upregulated IL4 mRNA expression. However phorbol ester and calcium ionophore did not affect the mean level of IL4 production by either B-CLL or normal B-cells. Furthermore, in the presence or absence of activation, the amount of IL4 synthesised by B-CLL B-cells was not significantly different than that observed with peripheral blood B-cells isolated from normal individuals (with activation: P=0.239; without activation: P=0.565). Also, there was no significant difference between normal and B-CLL B-cells in the level of cytoplasmic IL4 (P=0.47). PHA-activated enriched B-CLL T-cells produced significantly higher levels of IL4 compared to normal control T-cells (P=0.0136). In addition, in 47% of cases with B-CLL T-cells, a significant higher level of intracellular IL4 was observed (P=0.0027). The levels of production of IL4 by the T-cell-enriched preparations correlated positively with the intensity of cytoplasmic L4 in CD4+ and CD8+ cells in tested samples (r=0.49 and r=0.76, respectively). The significant differences observed in the production of IL4 by B-CLL B- and T-lymphocytes may suggest a paracrine function of IL4 in B-CLL.

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.

Effect of anti-APO1 on spontaneous apoptosis of B cells in chronic lymphocytic leukaemia: the role of bcl-2 and interleukin 4.

The cell surface protein apolipoprotein 1 (APO1) is expressed on various cell types including malignant lymphoid cells. Triggering of APO1 protein with antibody (Ab) induces apoptosis in APO1-expressing cells. We examined the effect of anti (alpha) APO1 Ab on spontaneous apoptosis (SA) and bcl-2 expression in B cell chronic lymphocytic leukaemia (B-CLL) in vitro. We also investigated the anti-apoptotic activity of interleukin 4 (IL4) on the aAPO1-induced apoptosis in B-CLL cells. Although expression of APO1 on B-CLL cells was not detectable by immunofluorescence, alpha APO1 Ab induced apoptosis in these cells. At 24 hours in culture the number of apoptotic cells was increased by a mean percentage (%) of 27% (range: 21-38) in only half of the cases studied. But in all twelve cases studied, at 48 hours alpha APO1 increased SA by a mean of 72% (range: 26-114) (P < .001) and at 72 hours, the mean % increase was 69% (range: 31-96) in 6/7 cases (P < .001). This effect was alpha APO1 concentration dependent. Interleukin 4 significantly protected B-CLL cells against alpha APO1-induced apoptosis by a mean of 53% (range: 28-76) (P < .001). This protection was specific to IL4 and it was significantly reduced or abolished with alpha IL4 Ab. Expression of bcl-2 protein in untreated cultures was not significantly different from that of the alpha APO1-treated cells; the mean equivalent of soluble fluorochrome (MESF) (range) was 4.9 (3.0-6.8) and 5.2 (3.5-6.0) respectively (P > 0.2). In fresh B-CLL cells the MESF (range) was 4.5 (2.4-6.6). Thus alpha APO1 Ab induced apoptosis in B-CLL cells by a pathway that is independent of bcl-2 expression and partially blocked by IL4.

Tumour necrosis factor gene polymorphisms in lymphoproliferative disease.

Tumour necrosis factor (TNF) alpha is involved in the pathogenesis of established lymphoproliferative disease. Serum levels of TNFalpha and its soluble receptors are above normal values in B-cell chronic lymphocytic leukaemia (B-CLL) and they are valuable prognostic markers in lymphoma patients. The production of TNFalpha is genetically controlled. Altered synthesis of TNFalpha has been associated with polymorphisms at the TNF gene cluster (i.e. TNFA, TNFB and LTB). In the present study, we evaluated the prevalence of the known high TNFalpha- and TNFbeta- producing alleles TNF1, TNF2 of the TNFA gene, TNFB1, TNFB2 alleles of the TNFB gene and of the polymorphic alleles TNFd1, d2, d3, d4 and d5 of the microsatellite TNFd in patients with B-CLL, non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). This study demonstrates that there is no difference in the frequency of the tested TNF alleles between normal controls and cohorts of patients with lymphoproliferative disease. These results indicate that TNF alleles are not genetic predisposing factors in the development of these diseases.

Effect of iron deficiency on the response of mouse lymphocytes to concanavalin A: the importance of transferrin-bound iron.

The in vitro response to Con A of lymphocytes from iron-deficient and normal mice in media containing either 10% fetal calf serum, apotransferrin or 20% iron-saturated transferrin was similar for the iron-deficient and control groups. However, the degree of proliferation in serum-free medium containing apotransferrin was significantly lower in all groups, compared to the responses in media containing either 20% iron-saturated transferrin or 10% fetal calf serum. Proliferation of lymphocytes from normal, iron-deficient or iron-repleted mice was lower in media supplemented with serum from iron-deficient mice than when serum from normal or iron-repleted mice was used. Addition of sufficient iron to bring the iron level of the deficient serum to that of normal serum significantly improved its ability to promote proliferation, while in vivo repletion of iron-deficient mice resulted in a restoration of normal lymphocyte responses to Con A. The proportion of cells positive for Thy 1.2, Ly 1 and Ly 2 antigens did not differ significantly between any groups of mice. Protein synthesis by cells proliferating in serum-free medium containing apotransferrin or 20% iron-saturated transferrin was the same in all groups of mice. These results indicate that decreased lymphocyte proliferative responses in iron deficiency may be due to inadequate levels of circulating transferrin-bound iron, rather than to intrinsic defects in the cells themselves or changes in the proportions of different T-cell subsets, and that iron availability does not affect protein synthesis by proliferating lymphocytes.

Tumour-associated proliferative responses in vitro of regional lymph nodes draining solid cancers in man.

The proliferative responses in vitro of tumour-draining lymph node lymphocytes were evaluated against autologous colon and lung carcinoma cells. The reactivity of lymphocytes appeared to be directed against tumour-associated rather than tumour-specific antigens. The lymphocyte reactivity detected was not due to an autologous mixed lymphocyte reaction. Recombinant interleukin-2 augmented the responses detected but not their tumour specificity. Phenotypic characterisation indicated the presence of T suppressor/cytotoxic (TS/C) cells as well as natural killer (NK) cells. Only the later, however, were active in functional cytotoxicity assays. The inability to generate both tumour-specific proliferation of tumour-draining lymph node lymphocytes and tumour-specific cytotoxic killer cells may be due to the presence of suppressor cells in the regional lymph nodes; preliminary data suggest the presence of such cells.

The role of iron and transferrin in lymphocyte transformation.

It has recently become apparent that many types of transformed cells possess membrane receptors for the plasma iron-binding protein transferrin. In this review Jeremy Brock and Tryfonia Mainou-Fowler discuss these observations in relation to transferrin's properties as an iron-transport protein, and the relative importance of iron and transferrin in lymphocyte transformation.

Evidence that transferrin may function exclusively as an iron donor in promoting lymphocyte proliferation.

In order to distinguish between a requirement for iron and a possible additional requirement for the iron-binding protein transferrin per se, the ability of mouse lymphocytes to proliferate in response to concanavalin A has been investigated. Cells proliferated well when cultured in medium containing 5% fetal calf serum, but if iron-free mouse or human transferrins were added, proliferation was inhibited by greater than 80%, whereas the same transferrins saturated to 30% with iron enhanced proliferation by 40-70%. In serum-free medium, proliferation was greater in the presence of 30% iron-saturated transferrin than when the protein was saturated only to 10%. Addition of Mn3+ to the latter, to bring the total metal saturation to 30%, gave no improvement in proliferation. Lymphocytes took up iron preferentially when transferrin containing both iron and manganese was present in the culture medium. The degree of proliferation in serum-free medium in the presence of a variant of human transferrin with abnormal iron-binding and receptor-binding properties was almost identical to that when normal human transferrin was used. Finally, when a monoclonal antibody to the mouse transferrin receptor and iron-nitriotriacetate were substituted for iron-transferrin in serum-free medium, proliferation was reduced by greater than 95%. These results strongly suggest that transferrin promotes lymphocyte proliferation solely as a result of its iron-donating properties, and that an additional role such as the provision of a proliferation-inducing membrane signalling event following interaction with the transferrin receptor seems unlikely.

Interleukin-5 (IL-5) increases spontaneous apoptosis of B-cell chronic lymphocytic leukemia cells in vitro independently of bcl-2 expression and is inhibited by IL-4.

During hematopoiesis, viability factors that suppress apoptosis are required throughout the differentiation process. Some of these factors may also function as growth factors. Interleukin-5 (IL-5) is recognized as a growth factor in hematopoiesis. We examined the involvement of IL-5 as a viability factor of B-CLL in vitro. In 13 B-CLL cases studied, IL-5 at 20 U/mL increased spontaneous apoptosis by a mean percentage of 53% (range, 20% to 129%) (P < .05) after 2 days in culture. On the third day, the mean percentage increase was 37% (range, 18% to 50%). In all cases, IL-4 protected B-CLL cells against IL-5-induced apoptosis by a mean percentage of 47% (range, 18% to 81%) (P < .001). This protection was specific to IL-4 and it was reduced with anti-IL-4 antibody. In addition, expression of bcl-2 protein in untreated cultures was not significantly different from that of the IL-5-treated cells; mean equivalent of soluble fluorochrome (MESF) was 5.2 (range, 3.0 to 6.8) and 4.9 (range, 3.0 to 6.3), respectively (P > .2). In freshly isolated B-CLL cells, the MESF was 4.5 (range, 2.4 to 6.6). These results show that IL-5 induced apoptosis in B-CLL cells by a pathway that is independent of bcl-2 expression. IL-4 partially protects against this effect.