Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells

Tumoral lymphocytes from patients with B-chronic lymphocyticleukemia (B-CLL) are long-lived cells in vivo, but they die rapidly byapoptosis in vitro. Here, it is reported that endothelial cells (ECs)inhibit the apoptosis of B-CLL cells, as determined by 4 different flowcytometric methods, and that this antiapoptotic effect is mediatedmainly by soluble factor(s), as can be deduced from the followingfindings. First, EC-conditioned medium (ECCM) inhibited the apoptoticrate in B-CLL to approximately 50% of control. Second, theantiapoptotic effect mediated by EC/B-CLL cell contact was moreapparent than real; using a fluorescence-based phagocytosis assay, itwas demonstrated that this effect was due to the phagocytic capacity ofECs, which internalized apoptotic cells. Third, the protective effectof ECCM was associated neither with proliferation nor differentiationsignals. Fourth, the survival factor was a dimeric form of IL-6 becauseanti-IL-6 antibodies completely neutralized the antiapoptotic effectmediated not only by the crude ECCM but also by the 45- to 55-kd activefractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6_D) were noncovalentlyassociated. Sixth, human recombinant IL-6_D(hrIL-6_D) inhibited B-CLL apoptosis, whereas hrIL-6monomers (hrIL-6_M) did not. Binding and functional competition experiments showed not only that monomers and dimers hadsimilar affinity for the IL-6R, but also that hrIL-6_Minhibited the antiapoptotic activity of hrIL-6_D. These datasuggest that IL-6_D derived from ECs promote the survival ofB-CLL cells.     

Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells.

The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)-alpha and PMA. The concentration of PMA was found to be critical and showed a sharp optimum. In most cases maximal proliferation was obtained with as little as 0.1 ng/mL PMA. In all cases tested, TNF-alpha-induced proliferation could be inhibited completely by the addition of low doses of interleukin-4 (IL-4). Maximal inhibition was already found with 400 pg/mL IL-4. Inhibition by IL-4 was not caused by a downmodulation of TNF receptors. Apart from TNF-alpha, IL-2 was also in synergy with PMA able to induce proliferation in B-CLL cells of some patients. This IL-2-induced proliferation could be inhibited both by IL-4 and by neutralizing anti-TNF-alpha antibodies. This shows that TNF-alpha also can act as an autocrine growth factor. These data indicate that TNF-alpha is an important growth factor for neoplastic B-CLL cells and that IL-4 provides a tool to interfere with this TNF-alpha response.     

Cell cycle progression of B-chronic lymphocytic leukemia cells induced to differentiate by TPA

The cell cycle transition and differentiation-associated surface antigen expression was studied in a clone of B cell chronic lymphocytic leukemia (B-CLL) with phenotypic properties similar to those of resting B lymphocytes. Differentiation was induced with TPA (12-O-tetradecanoyl- phorbol-13-acetate) and defined and quantitated by morphological and functional markers. Changes in the cell cycle position were determined by flow cytometry of acridine orange-stained cells. The uninduced B-CLL cells represented a homogeneous population with the same cell cycle position (GO) as resting normal peripheral blood lymphocytes. After five days of TPA stimulation, 56% of the B-CLL cells were found in G1A, 9% in G1B, and 3% in the S + G2/M phase, of which 2% was accounted to proliferating T cells. The cell cycle transition of the differentiating B-CLL cells was also examined using cell cycle-associated surface antigens as markers. HLA-DR and CD23 antigens were present already on noninduced cells. The former had a high constant expression, while the amount of CD23 increased upon induction. The 4F2 antigen was absent on noninduced cells but present on 86% of the induced cells. HH1 (CD37) was expressed by the majority of the cells before TPA treatment and decreased to almost undetectable levels within 24 hours. Two antigens related to late stages of the cell cycle, the interleukin 2 (IL 2; CD25) and the transferrin receptor, were present on about 20% of the induced cells. Experiments with enriched T cells showed that T but not B cells incorporated 3H-thymidine. Taken together these results and previous work on the induction of the protooncogene c-myc and c-fos suggest that this B-CLL clone represents GO cells that undergo differentiation without concomitant proliferation when exposed to TPA.     

Expression of functional toll-like receptors by B-chronic lymphocytic leukemia cells

This study reports that B-chronic lymphocytic leukemia (B-CLL) cells display the same pattern of toll-like receptors (TLRs) proteins expression as normal B-cells, yet with overexpression of TLR9. Furthermore, TLR7 and TLR9 appear to be functional and liable to respond to specific ligands, respectively imidazoquinolines and CpG-ODN thus potentially opening new therapeutic approaches.     

Apoptosis or plasma cell differentiation of CD38-positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman delta-chain antibodies (Gadelta-ab) caused [Ca(++)]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman mu-chain antibody (Gamu-ab) treatment in vitro. However, Gadelta-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gadelta-ab did not prevent apoptosis of B-CLL cells induced by Gamu-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL. (Blood. 2000;95:1199-1206)     

Interleukin-6 and tumor necrosis factor-alpha gene polymorphisms in chronic idiopathic urticaria

BackgroundThis study was performed to evaluate association of gene polymorphisms among proinflammatory cytokines and susceptibility to chronic idiopathic urticaria (CIU).MethodsNinety patients with prolonged urticaria more than 6 weeks were included as case group. Single nucleotide polymorphisms (SNPs) of IL-6 (G/C −174, G/A nt565) and TNF-α (G/A −308, G/A −238) were evaluated, using polymerase chain reaction (PCR); and the results were compared to the control group.ResultsG allele was significantly higher in the patients at locus of −238 of promoter of TNF-α gene (p < 0.001). Frequency of following genotypes were significantly lower in patients with CIU, compared to controls: AG at −308 and GA at −238 of TNF-α gene (p < 0.05 and p < 0.001, respectively), CG at −174 and GG at +565 of IL-6 gene (p < 0.05). Additionally, following genotypes were more common among patients with CIU: GG at −308 and −238 of TNF-α gene (p < 0.05 and p < 0.001, respectively), GG at −174 and GA at +565 of IL-6 gene (p < 0.05).ConclusionsPro-inflammatory cytokine gene polymorphisms can affect susceptibility to CIU. TNF-α promoter polymorphisms as well as IL-6 gene polymorphisms are associated with CIU.     

Bryostatin 1 induces differentiation of B-chronic lymphocytic leukemia cells

Peripheral blood cells from nine patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate). Like the phorbol ester 12-0-tetradecanoyl-phorbol 13-acetate (TPA), bryostatin 1 activates protein kinase C (PKC), which plays a central role in the phosphatidylinositol signal transduction pathway. The effects of bryostatin 1 alone and in combination with TPA or with the calcium mobilizing ionophore A23187 were assessed by morphological appearance, cell adherence and aggregation, RNA and DNA synthesis, and immunoglobulin (Ig) production. While eight of nine B-CLL cultures remained proliferatively inert, bryostatin 1 could effectively trigger activation and differentiation of B-CLL cells in all cases as inferred by the induction of morphological changes, RNA synthesis, and monotypic Ig production. Addition of calcium ionophore A23187 to bryostatin 1-exposed cells resulted in significantly increased values for RNA synthesis and Ig production and in the acquisition of plasmacytoid morphology. Bryostatin 1 and the dual signal of bryostatin 1 plus A23187 mimicked the stimulatory action of TPA and the combination of TPA plus A23187, respectively. Overall, bryostatin 1 was less active than equivalent concentrations of TPA. This lesser efficacy may, however, reflect a quantitative rather than qualitative difference. Bryostatin 1 partially antagonized TPA-mediated effects on B-CLL cells suggesting different modes of action by the two activators. These studies indicate that bryostatin 1 has effective differentiation-inducing properties on B-CLL cells that can differentiation-inducing properties on B-CLL cells that can be accentuated by a calcium ionophore.     

Heterogeneous proliferative effect of tumor necrosis factor-α and lymphotoxin on mitogen-activated B cells from B-chronic lymphocytic leukemia

The proliferative effect of tumor necrosis factor-α (TNF-α) and lymphotoxin on B cells from patients with B-chronic lymphocytic leukemia (B-CLL) was studied. Fresh purified B-CLL lymphocytes showed no proliferative response to either recombinant (r) TNF-α or r-lymphotoxin. However, after 'in vitro' activation of B-CLL lymphocytes for 2 days with Staphylococcus aureus Cowan 1 (SAC), four of seven patients showed enhanced blastogenic response in the presence of either rTNF-α or r-Iymphotoxin. We also found that the proliferative response of SAC-activated B-CLL lymphocytes to the two cytokines was independent of that found in the presence of interleukin-2. These results demonstrate that TNF-α and lymphotoxin can heterogeneously support the proliferation of in vitro activated B cells from B-CLL patients and may reflect the biological heterogeneity of B-CLL disease.     

Interleukin-6 and tumor necrosis factor-alpha in patients with obstructive sleep apnea-hypopnea syndrome

BACKGROUND: In previous studies, significantly elevated levels of vascular endothelial growth factor (VEGF) have been reported in patients with severe obstructive sleep apnea-hypopnea syndrome (OSAHS). On the other hand, plasma tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been significantly higher in mild sleep apneics than in normal controls. However, this study included a small number of patients and milder cases of OSAHS. OBJECTIVES AND METHODS: To assess the involvement of IL-6 and TNF-alpha in VEGF increases in patients with severe OSAHS, serum levels of IL-6 and TNF-alpha were determined in patients with severe OSAHS (n=110) and compared to those of controls (n=45) using an enzyme-linked immunosorbent assay. RESULTS: No significant increase in IL-6 or TNF-alpha was detected in the present study cohort. However, the body mass index was significantly correlated with the severity of the apnea-hypopnea index. CONCLUSIONS: These data suggest that the elevation in VEGF is not directly related to IL-6 or TNF-alpha levels. However, the question of whether VEGF is the cause or the result of OSAHS remains to be determined. Further studies are needed to clarify the role of IL-6 and TNF-alpha in the pathogenesis of OSAHS, in which obesity should be entered as an independent factor.     

Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis

Human interleukin-13 (IL-13) acts at different stages of the normal B- cell maturation pathway with a spectrum of biologic activities overlapping those of IL-4. B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells, arrested at the intermediate stage of their differentiation. In vitro, B-CLL cells exhibit a spontaneous apoptosis regulated by different cytokines. In this report, we show that IL-13 (10 to 200 ng/mL) acts directly on monoclonal B-CLL cells from 12 patients. (1) IL-13 enhances CD23 expression and induces soluble CD23 secretion by B-CLL cells but does not exhibit a growth factor activity. (2) IL-13 inhibits IL-2 responsiveness of B-CLL cells, activated either with IL-2 alone or through crosslinking of lgs or ligation of CD40 antigen. (3) IL-13 protects B-CLL cells from in vitro spontaneous apoptosis. The effects of IL-13 on neoplasic B cells were slightly less than those of IL-4 and occurred independently of the presence of IL-4. The present observations show that IL-13 may exhibit a negative regulatory effect on neoplasic B cells in contrast with that observed in normal B cells, and suggest that IL-13 could be an important factor in the pathogenesis of CLL by preventing the death of monoclonal B cells. Moreover, B-CLL may be an interesting model to study the regulation of the expression of IL-13 receptor and/or signal transduction pathways.     

Mechanisms of pulmonary edema induced by tumor necrosis factor-alpha

We tested the hypothesis that human recombinant tumor necrosis factor-alpha (TNF) promotes pulmonary edema by neutrophil-dependent effects on the pulmonary vasculature. The isolated guinea pig lung was perfused with phosphate-buffered Ringer's solution with or without human neutrophils. The infusion of neutrophils (9 x 10(6) total) into lungs isolated after the in vivo administration of TNF (3.2 x 10(5) units/kg) resulted in weight gain (+1.951 +/- 0.311 g versus -0.053 +/- 0.053 g in control) and an increase in the lung (wet-dry)-to-dry weight ratio (8.3 +/- 0.5 versus 6.0 +/- 0.2 in control), indicating the formation of pulmonary edema. The neutrophil-dependent pulmonary edema induced by TNF was associated with a combination of increased capillary permeability (capillary filtration coefficient [Kf,c], 0.170 +/- 0.048 g/min/cm H2O/g at 30 minutes versus 0.118 +/- 0.008 g/min/cm H2O/g at baseline) and increased pulmonary capillary pressure (Ppc, 12.8 +/- 0.8 cm H2O at 60 minutes versus 6.0 +/- 0.3 cm H2O at baseline). The Ppc increase was mediated by thromboxane A2 (TXA2) because the TXA2 synthetase inhibitor Dazoxiben (0.5 mM) prevented the effect (Ppc, 6.7 +/- 0.6 cm H2O at 60 minutes with Dazoxiben), and thromboxane B2 (TXB2) levels were increased in the pulmonary venous effluent (5,244 +/- 599 pg/ml at 60 minutes versus 60 +/- 13 pg/ml at baseline). Studies using WEB-2086 (37 microM), a platelet activating factor (PAF) receptor antagonist, indicated that PAF mediated the increased vascular permeability (Kf,c, 0.107 +/- 0.014 g/min/cm H2O/g at 30 minutes using WEB-2086) and, in part, the increased Ppc (Ppc, 8.4 +/- 0.7 cm H2O at 60 minutes using WEB-2086). In addition, alterations of endothelial peripheral actin bands were noted after TNF administration. The data indicate that TNF induces neutrophil-dependent pulmonary edema associated with increased Ppc (mediated by TXA2 and PAF), increased Kf,c (mediated by PAF), and changes in endothelial peripheral actin bands.     

The clinical significance of tumor necrosis factor-alpha plasma level in patients having chronic lymphocytic leukemia

Tumor necrosis factor-alpha (TNF-alpha), a cytokine possessing pleiotropic biological activities, is produced by leukemic lymphocytes in patients with chronic lymphocytic leukemia (CLL) and acts as an autocrine and paracrine growth factor in this disease. In this study, TNF-alpha levels were determined in 150 patients with CLL and correlated with disease characteristics, prognostic factors, and survival. The mean TNF-alpha plasma concentration in the patients with CLL was significantly higher than in the healthy control population (16.4 versus 8.7 pg/mL; P <.0001). Patients having an elevated TNF-alpha level had more advanced Rai and Binet stage disease, higher serum beta(2)-microglobulin (beta(2)M) levels, a greater percentage of cells expressing CD38, and lower hemoglobin and platelet levels. Patients having chromosomal abnormalities such as 11q deletion, trisomy 12, and chromosome 17 aberrations had a higher mean TNF-alpha level (27.5 pg/mL) than patients having a diploid karyotype or other miscellaneous cytogenetic abnormalities (14.2 pg/mL; P <.001). The TNF-alpha level was a predictor of survival when the Cox proportional hazards model was used with TNF-alpha entered as a continuous variable (P =.0001). Also, patients having a TNF-alpha level above the mean value of 14 pg/mL had significantly shorter survival duration (P =.00001). The TNF-alpha level remained predictive of survival in Cox multivariate analysis independent of Rai staging and beta(2)M, hemoglobin, prior therapy, white cell count, and platelet level (P =.005). We conclude that the TNF-alpha level serves as a prognostic factor in patients with CLL and that inhibition of TNF-alpha in these patients could have therapeutic importance.     

Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α

AIM： To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-α （TNF-α） on intestinal epithelial cells. METHODS： Caco-2 cells were incubated with TNF-α alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 （IL-8） secretion by intestinal epithelial cells was measured using an ELISA. Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase （ERK）, anti-phospho- ERK and anti-IκB-α. RESULTS： A TNF-α-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF- α-induced IL-8 secretion was reduced by L. plantarum. L. plantarum inhibited the activation of ERK and the degradation of IκB-α in TNF-a-treated Caco-2 cells. CONCLUSION： Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-α is inhibited byL. plantarum. Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.     

Production of tumor necrosis factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease

Tumor necrosis factor-alpha (TNF) is a cytokine that displays a pleomorphic array of effects on different cell populations. Evidence is presented that TNF may be constitutively produced by B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells and that it may play a relevant role in these diseases. These conclusions are based on the presence of circulating levels of TNF in the serum of 20 of the 24 patients tested (83.3%), while undetectable values were found in normal sera. The suggestion that the increased serum levels were due to the leukemic cell population is strengthened by the evidence that purified B-CLL and HCL cells may constitutively release variable degrees of TNF. These levels markedly increase after incubation with interferon gamma or phytohemagglutinin (PHA) plus phorbol myristate acetate (PMA). The cellular release of TNF by primary B-CLL cells was significantly (P less than .001) higher in B-CLL stage O-I patients compared with stage II-III patients. The demonstration that, in B-cell chronic lymphoproliferative disorders, the pathologic cells may release TNF was further confirmed by the presence of the mRNA for this cytokine in primary and/or in pre-activated cells. Recombinant TNF was capable of inducing a proliferative signal only in a minority of cases (4/24); in most cases it was ineffective, and, in a few, it reduced the degree of proliferation. Furthermore, in costimulatory experiments with interleukin-2 and PHA plus PMA, TNF was ineffective. On the other hand, when primary B-CLL cells were incubated in the presence of an anti-TNF antibody, in 8 of 12 independent experiments a 2- to 15-fold increase in thymidine uptake was documented. Taken together, these results suggest that TNF may play a regulatory role in the progression of the neoplastic clone in B-cell chronic lymphoproliferative disorders and may be implicated in some of the side effects associated with these diseases.     

Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy. METHODS: RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay. RESULTS: Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate. CONCLUSION: Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.     

Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro

Summary. When monoclonal B cells from B-chronic lymphocytic leukaemia (B-CLL) patients are cultured in vitro , they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B-CLL patients after 26–30 h in in vitro culture, with 14.3–59.0% (mean 33.6%) of their DNA being fragmented in ∼180 base pair multimers. After 8–10 d culture, 90–100% of the B-CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5–5 ng/ml human recombinant interleukin-4 (IL-4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml. IL-4 reduced DNA fragmentation after a 26–30 h culture to 2.2–33.3% (mean 14.9%). IL-4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin-D. Western blot analysis of cell lysates showed expression of the bcl-2 protein in all 11 B-CLL patients studied. However, during culture, bcl-2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl-2 protein levels was inhibited in cells cultured in the presence of IL-4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B-CLL cells and indicate that IL-4 may participate in the extended survival of these non-dividing cells in vivo .     

Antigen modulation followed by quantitative flow cytometry of B-chronic lymphocytic leukemia cells after treatment

Presented study analyzed the immunophenotypic characteristics and antigen density of peripheral blood (PB) and bone marrow (BM) cells of 23 patients with B-chronic lymphocytic leukemia (B-CLL) and 10 control subjects using flow cytometry. The patients were subclassified into two groups. Group I formed 13 patients with B-CLL at the time of diagnosis and group II 10 patients with B-CLL after the therapy but not in remission. For definition of B-CLL cells we used immunological marker analysis of surface markers characteristic for B-CLL pattern: CD5, CD19, CD20, CD23 and HLA DR and enumeration of fluorescence intensity of these markers given by molecular equivalent of soluble fluorochrome--MESF. In group II of B-CLL patients, who were already treated, in PB and BM somehow lower MESF values for CD19, CD20 and CD23 markers and higher MESF values for CD5 marker (in PB and BM) than in group I patients have been detected. The MESF level of HLA DR marker was little higher in group II than in group I B-CLL patients. However in PB and BM the percentage expression of HLA DR and CD19 markers in both patients groups was approximately the same. The values of HLA DR, CD19, CD20, CD23 and CD5 markers (% expression and MESF values) in both patients groups with B-CLL were significantly higher versus controls (p<0.001 resp. p<0.01) even in PB and BM. In conclusion, in our study we observed that the patients with B-CLL after therapy (group II) had similar or a little smaller (except CD5) but nonsignificantly decreased expression level of markers characteristic for B-CLL, but the MESF values of some of them (CD19, CD23) were significantly (p<0.05) decreased when compared with untreated B-CLL patients (group I). The determination of antigen density (MESF values) may be an important marker to characterize the leukemic cells. Our results showed that chemotherapy did not influence in a significant level the antigen modulation of B-CLL cells, however, could influence MESF values of some characteristic markers. Quantitative analysis of some markers in B-CLL cells seems to offer valuable information concerning possible influence of some chemotherapeutics on antigen equipment of leukemic cells.