Serum levels of TNF, IL-6 and sCD23 correlate with changes in lymphocyte count in patients with B-cell chronic lymphocytic leukaemia receiving interferon-alpha therapy.

The control of cell growth and differentiation in B-cell malignancies may be regulated by the autocrine production of cytokines, several of which have been implicated in the growth and survival of B-cells. The effect of interferon-alpha (IFN) therapy in these disorders may be to disrupt autocrine growth or survival loops. We have measured levels of circulating IL-1b, IL-6, TNF-a and soluble CD23 (sCD23) in 8 patients with Binet stage A B-cell chronic lymphocytic leukaemia (B-CLL) receiving IFN therapy, and compared these with changes in the lymphocyte count following IFN therapy. Two patients developed anti-interferon antibodies while on IFN therapy, and in both them, the changes in lymphocyte count correlated significantly with the titre of anti-interferon antibodies, as well as serum levels of IL-6, TNF-a and sCD23. In one patient there was significant correlation with levels of IL-1b. One patient, who stopped and restarted IFN therapy, demonstrated correlation between lymphocyte count and levels of IL-6 and sCD23. In a further two patients, there was correlation with levels of sCD23 alone, while the remaining three patients showed no correlation between lymphocyte count and the serum cytokines measured. These results suggest that IFN therapy may alter levels of circulating cytokines in some CLL patients and that these effects may be associated with disease progression.

Growth factors can protect B-chronic lymphocytic leukaemia cells against programmed cell death without stimulating proliferation.

The proliferation and survival of B-chronic lymphocytic leukaemia (B-CLL) cells may be regulated by autocrine growth factor loops. Furthermore, it has been suggested the reduction in lymphocytosis following therapy with interferon-alpha may be associated with the interruption of autocrine growth factor production. We have therefore examined the effects of a number of cytokines on the proliferation of B-CLL cells, and also on the regulation of programmed cell death, and the role of interferon-alpha in these systems. In the ten patients studied, neither interferon-alpha alone or together with either interferon-gamma, IL1, IL4, IL6, TNF, or serum containing high levels of soluble CD23 was able to induce proliferation of B-CLL cells. Incubation with TPA or IL2 resulted in variable proliferative responses. Co-incubation with interferon-alpha enhanced TPA-induced proliferation in 4 cases, but reduced IL2-induced proliferation in all cases studied. In contrast, all the cytokines studied were able to protect B-CLL cells against programmed cell death, both spontaneous and that induced by hydrocortisone, with the exception of TNF. These data suggest a role for interferon-alpha in disrupting autocrine survival pathways rather than inhibiting proliferation.

BLT-esterase in infectious mononucleosis.

Peripheral blood lymphocytes of three patients suffering from infectious mononucleosis due to Epstein-Barr virus (EBV) infection were analysed for BLT-esterase expression in peripheral blood lymphocytes by a well established cytochemical staining method. During the acute phase of disease with presence of clinical symptoms a very high level of up to 90% BLT-esterase-expressing lymphocytes were detected. The increased percentage of lymphocytes expressing BLT-esterase coincided with the time of greatest symptoms and the peak elevation of hepatocellular enzymes. The still moderately elevated level only gradually decreased to normal during the further recovery period of 2 months during which the patients described episodes of weakness. Peripheral blood lymphocyte phenotype analysis revealed a marked CD8 lymphocytosis, a CD4/CD8 ratio of about 0.2, low number of CD19+ B cells, and a high level of DR+ CD3+ lymphocytes. Reduction of BLT esterase expression during the recovery period coincided with reduction of CD8+ DR+ lymphocytes. By a combination of BLT-esterase staining with immunocytochemical phenotype analysis, 95% of CD8+ lymphocytes were found to be BLT-esterase-positive. BLT-esterase might be involved in the immunodefence against EBV in infectious mononucleosis by inducing apoptosis in EBV-transformed B cells.

Changes in maternal plasma macrophage-colony stimulating factor levels during normal pregnancy.

An enzyme-like immunosorbent assay and a blood autoanalyser were used to determine macrophage-colony stimulating factor (M-CSF) levels and the absolute number and percentage of circulating monocytes in 80 normal women with singleton pregnancies at 12-40 weeks' gestation, and ten healthy non-pregnant volunteers. The mean values of M-CSF and absolute number and percentage of circulating monocytes of the control group were 367 U/ml (SD 43) and 389 x 10(6)/l (SD 180) and 5.3% (SD 1.7) respectively. In pregnancy, M-CSF was significantly higher than non-pregnant controls only after 28 weeks' gestation. The absolute number and the percentage of circulating monocytes increased significantly with gestation after 16 weeks. There was no significant association between the concentration of M-CSF and the number or percentage of circulating monocytes. These data suggest that during pregnancy there is an up-regulation of M-CSF and monocytes.

Myeloperoxidase activity and nuclear segmentation of maternal neutrophils during normal pregnancy.

Myeloperoxidase (MPO) activity and nuclear segmentation of neutrophils were measured in peripheral blood samples from 90 normal pregnant women, arranged in nine groups of ten each, every four weeks at 8-40 weeks gestation and from 25 non-pregnant healthy female controls. A blood autoanalyser (Technicon H*1) was used to determine the mean peroxidase index (MPXI) and the lobularity index (LI) of circulating neutrophils. Mean MPXI levels in pregnancy decreased with gestation to a minimum at 20 weeks' gestation and increased thereafter to reach non-pregnant levels at 36 weeks. Mean LI values did not change significantly with gestation, but were significantly lower throughout pregnancy compared to controls. There was no significant association between MPXI and LI. It could be postulated that the fall in MPXI during mid-gestation is due to degranulation of neutrophils and the subsequent rise at the end of pregnancy could be a consequence of enhanced MPO activity under the influence of oestrogens. Low LI suggests the production of immature neutrophils during pregnancy.

Interferon-alpha up-regulates bcl-2 expression and protects B-CLL cells from apoptosis in vitro and in vivo.

The bcl-2 oncoprotein, which is involved in the t(14,18) translocation, protects cells against apoptosis. We examined the effects of interferon-alpha (IFN-alpha) on bcl-2 protein expression and apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. None of 12 patients with B-CLL examined expressed the t(14,18) translocation; however, all these, and seven other patients, expressed significant levels of bcl-2 protein. In vitro, IFN-alpha (500 U/ml over 18 h) increased bcl-2 expression on CLL cells (to 200 +/- 23% of control MCF, as determined by indirect immunofluorescence and flow cytometry, n = 10, P < 0.001). All of eight patients who received IFN-alpha (3 megaunits subcutaneously three times a week) demonstrated an increase in bcl-2 expression on circulating malignant cells. CLL cells undergo apoptotic cell death when cultured in vitro (35.6 +/- 10.3% DNA fragmentation after 18 h, n = 10). In the presence of IFN-alpha, however, DNA fragmentation was reduced to 6.6 +/- 5.8% (n = 10, P < 0.001). IFN-alpha also protected CLL cells against apoptosis induced by hydrocortisone and gamma irradiation (reducing DNA fragmentation from 63.9 +/- 12.6% to 10.8 +/- 4.5% and from 80 +/- 2.9% to 5.4 +/- 1.6%, respectively, P < 0.001 for both). The protective effect of IFN-alpha was dose dependent, and maintained for up to 24 h. Our data demonstrate that bcl-2 expression and apoptosis of CLL cells can be influenced by cytokines. In addition, it seems unlikely that the observed clinical responses to IFN-alpha in patients with CLL are due to a direct effect on the malignant cells.

Serum macrophage colony-stimulating factor (M-CSF) levels correlate with clinical response to interferon-alpha in patients with early-stage B-CLL.

Interferon-alpha (IFN-alpha) reduces peripheral lymphocyte counts in B-CLL (CLL). In eight patients with stage 0 CLL on IFN-alpha therapy, peripheral lymphocyte counts fell to 61.7 +/- 19.5% of baseline at week 2 (P < 0.01), while serum M-CSF levels rose from 455 +/- 183 U/ml to 686 +/- 110 U/ml (P < 0.05). Neopterin levels also showed a significant rise. M-CSF levels were correlated with clinical response in these patients. Increased production of M-CSF and the activation of mononuclear phagocytes may be involved in clinical responses to IFN-alpha in early-stage CLL.

Recombinant interferon alfa 2a in the treatment of patients with early stage B chronic lymphocytic leukaemia.

18 patients with early stage, previously untreated B-CLL were given interferon alfa (IFN alpha) 2a. 3 MU thrice weekly, subcutaneously. The peripheral lymphocyte count decreased in all patients. Response was delayed in three patients until they had received a median of 5 months therapy, one of whom had an initial transient increase in lymphocytes. Two patients normalized their blood lymphocyte counts, but neither achieved complete remission (CR). Responses were transient in eight patients lasting a median of 5 months (3-21). Binding anti-IFN alpha antibodies were present in 9/17 patients tested (53%). Low titre binding antibodies (< 533 IBU/ml) were not associated with LHR, but high titre antibodies (> 4401 IBU/ml) were. Two of 12 patients assessed had a > 3 g/l increase in baseline serum IgG levels during IFN alpha therapy, one of whom reverted to pretreatment levels in association with LHR. Haematological toxicity was moderate, other than in two patients, one of whom developed autoimmune haemolytic anaemia and the other thrombocytopenia. We conclude that IFN alpha lowers the lymphocyte count in early stage CLL, that the response may be delayed and that anti-IFN alpha antibodies may play a role in a proportion of those in whom the response is transient.

In vivo and in vitro primed lymphocytes. Correlation of cytochemically detected BLT-specific lymphoid serine protease with cytotoxic activity.

We describe the validation of a cytochemical method to detect a cytolytic cell-specific lymphoid serine protease which can be upregulated during viral infection and allogeneic stimulation. The cytolytic cell specificity was ascertained by demonstrating a high correlation between BLT substrate-specific serine protease (SP) activity and cytotoxicity of in vivo and in vitro stimulated lymphocytes. The presence of SP in peripheral blood lymphocytes was compared with their capacity to kill K562 targets in a lectin-dependent cytotoxicity assay. The correlation coefficient was 0.92 and 0.93 at E:T ratios 10:1 and 20:1 respectively. In allogeneic mixed lymphocyte cultures an increase of SP activity in effector lymphocytes was paralleled by an augmentation of cytotoxic capacity towards stimulator target cells. SP+ granules showed intracellular polarization to the effector/target cell interface during conjugate formation. These results together with previous studies suggest that this method provides a sensitive assay which predicts the cytolytic potential present in a lymphocyte population.

Low-dose recombinant alfa-2a-interferon: a feasible maintenance therapy in acute myeloid leukaemia in the older patient.

Eleven patients with acute myeloid leukaemia (AML) in first complete remission (CR) were treated with alfa-2a-interferon (for short 'interferon') maintenance therapy, at a dose of 3 MU twice to thrice weekly subcutaneously. Adjustments were made to maintain neutrophil counts > 1 x 10(9)/l and platelet counts > 100 x 10(9)/l. A transient fall in haemoglobin, neutrophil and platelet counts was noted in all 9 evaluable patients. Median time to nadir was 7 weeks. Initial dosage reductions were necessary in 5 patients, 3 of whom were later able to tolerate the starting dose. No episodes of infection or bleeding were documented during therapy and no red cell or platelet transfusions were necessary. At the time of writing (median follow-up of 31 weeks), 7 patients continue in CR, 6 of whom remain on interferon. One patient discontinued interferon on developing sicca syndrome. Other than in this patient, side effects were minor. Mean dose administered was 6.7 MU/patient/week. We conclude that low-dose IFN maintenance therapy is well tolerated in older patients with AML in first CR.

Resistance of chronic lymphocytic leukaemia cells to interferon-alpha generated lymphokine activated killer cells.

Recent studies have shown that, when used in early stage disease, interferon-alpha (IFN-alpha) can produce a fall in the number of malignant cells in the peripheral blood of patients with B-CLL. In this study, we investigated the effect of IFN-alpha on natural killer (NK) cell and lymphokine-activated cell (LAK) activity in patients with B-CLL. In vitro, IFN-alpha (500 U/ml for 18 hours) induced LAK activity in patients with B-CLL (27.7 +/- 9.9%, n = 20), and IL-2 (500 U/ml for 5 days) produced similar activity (35.9 +/- 8.8%, n = 7). Despite the induction of LAK activity by IFN-alpha and IL2 in patients with B-CLL, the malignant cells remained resistant to both allogeneic and autologous LAK effectors. NK activity in patients with B-CLL is also low (23.1 +/- 7.2%, n = 20), and B-CLL cells were resistant to NK cell activity. In cold target competition assays, CLL cells did not compete with labelled K562 or Daudi targets in the NK and LAK assays, suggesting that the malignant cells are not recognised by the effector cells, and this may be related to low level of expression of the adhesion receptors, LFA-1 and ICAM-1. Finally, CLL cells were also resistant to antibody dependent cell mediated cytotoxicity, but were susceptible to antibody dependent complement mediated lysis. These results suggest that it is unlikely that the effects of IFN-alpha in B-CLL are due to the enhancement of NK or LAK activity.

Cytokine induction of leucocyte adhesion molecule-1 (LAM- 1) expression on chronic lymphocytic leukaemia cells.

Leucocyte adhesion molecule 1 (LAM-1) participates in the binding of human leucocytes to high endothelial venules in peripheral lymph nodes. Other adhesion receptors which are involved include CD44 and the integrin family, CD11/CD18. In this study, B-cell chronic lymphocytic leukemia (B-CLL) cells were examined for the expression of these adhesion molecules, and for the way in which cytokines are able to modulate the levels of these receptors. B-CLL cells express significant but variable levels of LAM-1 and high levels of CD44. In contrast, these cells exhibit very low or absent amounts of surface CD11a, CD11b, or CD11c. Most CLL cells expressed no detectable levels of intercellular adhesion molecule-1 but some cases show levels of up to 30%. Following 24 h incubation with interferon alpha (500 U/ml), surface LAM-1 expression on peripheral blood E-negative cells from CLL patients rose to 330 +/- 127% of levels on control cells incubated with medium alone (n = 13, p less than 0.0005). Interleukin 4 (1 ng/ml) and interferon gamma (100 U/ml) also increased surface LAM-1 levels on these cells to 218 +/- 119% (n = 8, p less than 0.001) and 245 +/- 116% (n = 5, p less than 0.001) of control levels respectively. Induction of LAM-1 expression occurred over 48 h (greater than 50% of the increase was seen in the first 24 h) in a dose-dependent manner and required protein synthesis. The induction of LAM-1 expression on the malignant cells may, by altering the homing behaviour of these cells, relate to the reduction in peripheral leukaemic cells seen following treatment with interferon alpha in CLL.

Detection of BLT substrate-specific proteases in individual human peripheral blood leucocytes and bone marrow cells. Application of the method to the classification of leukaemia.

A trypsin-like serine esterase (SE) is known to be present in cultured cells with cytolytic potential. The distribution pattern of this enzyme in haematological cells and body tissues has been assessed using a method which permits rapid identification of individual cells. Cells and tissue sections were fixed and immersed in the substrate N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)/Fast Blue BB chromogen solution. To identify the phenotype of SE+ cells the cytochemical stain was followed by the application of monoclonal antibody and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex immunocytochemical procedures. CD8+ and CD57+ lymphocytes showed SE+ granules. Neutrophil granulocytes and progenitors other than undifferentiated myeloblasts developed a dense stain while eosinophils were negative. 35% of monocytes showed positivity mainly in the area of nuclear indentation. Tumour-infiltrating SE+ lymphocytes could also be demonstrated with this method.

Continuous intravenous infusion of high dose recombinant interleukin 2 for advanced lymphomas--a phase II study.

The prognosis of patients with advanced refractory lymphoma remains poor. We have carried out a Phase II study of continuous high dose intravenous recombinant interleukin 2 alone without LAK cells in this group of patients. Eight patients have so far been treated, 4 with non-Hodgkins lymphoma and 4 with Hodgkins disease. Of the 7 evaluable patients, the maximum response observed was stable disease in 2 patients (1 with NHL and 1 with HD). The other patients' diseases progressed in the face of immune activation following the rIL-2 infusion. Adverse reactions were common but no life-threatening occurred. These results are disappointing. Whether or not lymphokine-activated killer (LAK) cells are needed to improve response rate deserve further investigations.

Cellular cytotoxic function and potential in acute myelogenous leukaemia.

Twenty-seven adult AML patients (13 with active disease and 14 in complete remission) were investigated for their cellular cytotoxic potential and function. All AML patients, whether with active disease or in complete remission, showed increased percentage of CD3+ lymphocytes expressing the cytotoxicity-linked cytoplasmic serine esterase, suggesting a higher than normal cytotoxic potential. However, when the cytotoxic function in these patients were analysed in terms of the natural killer and lectin-dependent cellular cytotoxicity, all AML patients, whether with active disease or in complete remission, had impaired target cell lytic activity. This paradox of cytotoxicity is most likely due to the immunosuppressive effect of the serum factor elaborated by leukaemia myeloblasts.

Lymphocyte activation in patients with acute myeloid leukaemia. Evidence for the presence of myeloblast antigen?

A group of 27 patients with acute myeloid leukaemia (AML), 15 with active disease and 12 in complete remission, were investigated for evidence of T cell activation. The parameters of T cell activation measured were the serum levels of soluble interleukin-2 receptor (sIL-2R), soluble CD4 (sCD4) and soluble CD8 (sCD8) molecules and the proportions of T cells expressing the cytotoxicity-linked cytoplasmic serine esterase. All patients studied with active disease had elevated sIL-2R and sCD8 molecules and an elevated proportion of T cells expressing serine esterase. Patients studied in complete remission also had elevated sIL-2R. sCD8 and serine-esterase-positive T cells, but values were lower than those studied in active disease. These patients were all studied in the absence of any ongoing or recent infection or exposure to homologous blood products, either of which could potentially affect these parameters. In the absence of any obvious alternative cause, we suggest these data indicate that AML leukaemia blast cells may be immunogenic and lead to the activation of cytotoxic T cells.

Recombinant alpha-interferons, thyroid irradiation and thyroid disease.

Recombinant alpha-interferons are used as therapeutic agents in an increasing number of benign and malignant disorders. Long-term administration of recombinant alpha-interferon as a maintenance agent is associated with a small number of adverse side-effects which are responsible for patient intolerance of this drug. These include weight loss, alopecia and chronic fatigue, anorexia and depression syndrome. This latter syndrome needs to be distinguished from thyroid disease, which is documented in this report in a number of patients on recombinant alpha-interferon therapy.

Production of tumour-derived suppressor factor in patients with acute myeloid leukaemia.

In an attempt to investigate the underlying cause of impaired cellular cytotoxic functions in patients with acute myeloid leukaemia and the relative ineffectiveness of immunotherapy with recombinant interleukin 2 (IL-2), normal donor lymphocytes were incubated in AML sera and in supernatant of myeloblasts. There was significant inhibition of both the natural killer activity and the lectin dependent cellular cytotoxicity of the normal donor lymphocytes compared to when incubation took place in autologous or normal allogeneic sera or marrow supernatant. This inhibition was time-related and partially reversible by washing of the normal lymphocytes immediately before the cytotoxicity assay. The suppressor factor, however, did not inhibit the IL-2 induced lymphocyte proliferation or affect the cytotoxicity-linked cytoplasmic serine esterase expression in the normal lymphocytes. This suppressor phenomenon was of myeloblast origin. Chronic exposure to the tumour-derived suppressor factor may be responsible for the impaired cellular cytotoxic functions observed in patients with acute myeloid leukaemia. It may also suppress the in vivo cytotoxic functions of IL-2 activated lymphocytes in patients treated with recombinant IL-2, hence leading to the disappointing results of immunotherapy so often encountered in clinical setting.

Upregulation of a lymphoid serine protease in kidney allograft recipients.

The presence of a putative, cytotoxicity-linked lymphoid serine esterase (SE) has been studied in 79 kidney graft recipients. Peripheral blood lymphocytes (PBL) bearing an N-alpha-benzyloxy carbonyl-L-lysine thiobenzyl ester (BLT)-specific SE were evaluated by a novel cytochemical staining method. A characteristic of post-allograft patients was an increased presence of SE containing granules in PBL. In 46 patients with stable graft function SE + PBL were 33.41 +/- 10.34% (controls: 26.30 +/- 5.22%, P less than 0.0025), SE + CD4+ 4.32 +/- 3.85% (controls 2.13 +/- 1.52%, P less than 0.0025) and SE + CD8+ T cells 47.68 +/- 18.64% (controls: 28.50 +/- 6.50%, P less than 0.0005). In those graft recipients undergoing a rejection episode a marked upregulation of SE activity could be observed when compared to the stable graft group: SE + PBL were 59.91 +/- 10.89% (P less than 0.0005), SE + CD8+ 74.30 +/- 10.79% (P less than 0.0005) and SE + CD4+ T cells 28.56 +/- 13.50% (P less than 0.0005). In 10 cases this increase of SE activity was observed with a time lag of up to 37 days prior to the onset of clinical or biopsy proven rejections, promptly decreasing in response to methylprednisolone antirejection therapy. In patients with recurrent rejection episodes and subsequent graft loss, a repeating increase of SE activity indicated a failure of therapeutic agents.(ABSTRACT TRUNCATED AT 250 WORDS)