Quantification of the leukocyte common antigen (CD45) in mature B-cell malignancies.

CD45 is a glycoprotein expressed on all lymphohematopoietic cells. Its expression increases during normal B-cell differentiation and remains stable on mature cells. Although it is widely known that CD45 antigen expression is decreased in B-acute lymphocytic leukemia (ALL), only scarce and contradictory information is available on CD45 expression on mature B-cell malignancies. In healthy adults (n = 15), CD45 expression on B lymphocytes was lower than that on T cells. In patients with chronic lymphocytic leukemia (CLL; n = 22), CD45 expression on malignant cells was lower than that on the whole lymphocyte population of healthy adults (n = 28) and on normal B lymphocytes (n = 15). In 6 of the 22 CLL patients, the malignant cell population could be separated from the normal lymphocyte population on the CD45-side scatter (SSC) plot. In 16 CLL patients, there was some degree of overlap between the malignant and normal cells with respect to CD45 expression. For these patients, there was an inverse correlation between CD45 expression on the whole lymphocyte population and the percentage of malignant cells in this population. In two patients with mantle cell lymphoma (MCL), CD45 expression on the malignant cells appeared lower than that on normal B cells and on the whole lymphocyte population. In six patients with hairy cell leukemia (HCL), CD45 expression on hairy cells was comparable to that on the whole lymphocyte population of healthy adults, but slightly higher than that of normal B cells. Evaluation of CD45 expression may help to characterize mature B-cell malignancies.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

A review of the prognostic role of cytogenetic, phenotypic, morphologic, and immune function characteristics in chronic lymphocytic leukemia

Recent reports of successfully completed cytogenetic studies using polyclonal B-cell activators demonstrate that trisomy-12 and 14 q+ are the most frequently observed chromosomal abnormalities in B-cell chronic lymphocytic leukemia (CLL). It appears that when trisomy-12 is accompanied by yet another abnormality, the prognosis of patients is uniformly poor. Patients in early stages of CLL retain delayed hypersensitivity reactivity, while those in advanced stages are usually anergic. The lymphocytes from venous blood of patients with CLL appear to retain at least some ability to respond to stimulation with mitogens in early stages, whereas in advanced stages they show no response to mitogens. Serum immunoglobulin levels are normal in the early (0 and I) stages, are markedly decreased in the advanced (III and IV) stages, and are somewhat between these extremes in the intermediate (II) stage of CLL. Prolymphocytic leukemia and prolymphocytoid transformation of CLL are indicators of poor prognosis, while a morphological variant characterized by large granular lymphocyte is associated with good prognosis. At this time it is not possible to ascribe strong prognostic significance to phenotypic features of lymphocytes in B-CLL; however, studies currently in progress may soon provide important insights on this subject. We have reviewed the pertinent literature and we also present a summary of results from our laboratory.     

MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.     

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.     

In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

The binding of IgM to B lymphocytes: a comparison of the binding characteristics of IgM aggregates and EAB (IgM) complexes to normal and leukemic B lymphocytes

We have compared and contrasted the binding of Agg IgM and heavily sensitized EAB (IgM) complexes to the Fc mu receptor of normal and neoplastic human lymphocytes. Agg IgM binds uniformly to the entire SmIg+ B cell population yet normal lymphocytes require culture in order to achieve binding of EAB complexes to a subset of SmIg+ B cells. In blocking studies IgM complexes and IgM aggregates appear to detect the same receptor and with both reagents binding is influenced by the presence of Mg2+ but not Ca2+ and is inhibited by EDTA. The percentage of cells binding EAB was highest in normal B lymphocyte fractions enriched for C2+ cells (CRL+). EAB binding to cells in the CRL- fractions was negligible even though CRL- fractions contained cells which were SmIg+C-3. EAB bound only to neoplastic chronic lymphocytic leukemia cells (CLL) that expressed a high percentage of C+3 cells. Clones lacking a C3 receptor failed to bind EAB. Thus, the binding of EAB complexes to B lymphocytes appears to be associated principally with a subset that express a C3 receptor whereas IgM aggregates bind to the entire SmIg+ B cell population.     

Phorbol ester induces tyrosine phosphorylation in normal and abnormal human B lymphocytes

Tumor-promoting phorbol esters have been found to bind and activate phospholipid/Ca2+-dependent or C-kinase, and several of their effects, including proliferative responses in lymphocytes, have been assumed to be related to activity of this enzyme. However, phorbol esters have also recently been found to stimulate tyrosine phosphorylation in certain other cell types, and we therefore studied tyrosine kinase activity in normal and chronic lymphocytic leukemia (CLL) peripheral blood B lymphocytes stimulated with phorbol ester. High levels of tyrosine labeling were observed in unstimulated cells with major endogenous substrates of 75K, 66K, 43K, and 28K in Triton-soluble material, and of 56K to 61K in Triton-insoluble material; this profile was essentially similar in normal and CLL B cells. Treatment with phorbol ester for time periods varying from 20 min to 48 hr led to qualitative increases in tyrosine labeling of these phosphoproteins, as measured both in vitro and in intact cells "in vivo." Although the relative abundance of tyrosine phosphorylation as a percentage of total labeling was variable due to concomitant enhancement of serine and threonine phosphorylation, exogenous peptide substrate assays confirmed the increased tyrosine kinase activity quantitatively. Enhanced tyrosine phosphorylation was succeeded or accompanied in both normal and abnormal B cells by cellular activation, as judged by increased [3H]thymidine uptake, and terminal differentiation of CLL cells. These findings provide further evidence implicating tyrosine kinases in B lymphocyte activation.     

Does the CLL B lymphocyte correspond to expansion of an immature B clone or to expansion of a distinct subpopulation of B cells?

Most CLL cases correspond to proliferation of a B-cell clone characterized by (1) low amounts of SmIg, (2) the presence of receptors for mouse red blood cells, (3) the presence of a 67-kd antigenic determinant recognized by CD5 monoclonal antibodies, also present in normal T cells, (4) the ability of these cells to differentiate in vitro, and (5) the ability at least for some clones to differentiate and secrete immunoglobulins in vivo. The normal counterpart to this B-cell clone corresponds to a small subpopulation of lymphocytes, present at the edge of the germinal center in human lymph nodes. Interestingly, this subpopulation appears to constitute a substantial part of the B-cell population in 20-week-old fetal lymph nodes and spleen. These results have induced most authors to assume that the CLL B lymphocyte corresponds to proliferation of an immature B-cell clone, arrested at an intermediate stage between pre-B cells and mature B cells. However, this hypothesis does not explain the high frequency with which hypogammaglobulinemia and autoimmune hemolytic anemia are found in B CLL. In this work, we discuss the possibility that the CLL B lymphocyte corresponds to proliferation of a B-cell line, whose counterpart in the mouse is the Ly1 B, Lyb5+ subpopulation.     

Relationship between the capacity to be stimulated of lymphocyte subpopulations and the RAI staging in patients with chronic lymphocytic leukemia]

By means of the incorporation rate of 3H thymidine into the lymphocytes of patients with chronic lymphatic leukaemia the possibility of stimulating them by using different mitogens was checked and compared with normal persons. The examination covered 11 patients treated with extracorporeal irradiation of the blood (ECIB), 5 patients treated with a chlorambucil therapy, and 10 untreated patients who were classified according to the staging system proposed by RAI. The lymphocytes of the peripheral blood were stimulated as mixed and isolated T and B-lymphocytes in the microculture by using the mitogens PHA, PWM, ConA, and LPS. In all CLL patients there was a diminished stimulation rate of a mixed lymphocyte population. A relation existed between the seriousness of the stage and the diminution of the incorporation rate of 3H thymidine. A corresponding correlation could not be identified in untreated CLL patients. Isolated T-lymphocytes revealed better results of stimulation than the total population. As to their function B-lymphocytes showed a dependance on the kind of therapy. In the mixed lymphocyte culture of normal persons the best findings could be observed after stimulation with PHA, that is also valid for CLL patients. PHA, PWA, ConA, and LPS were suitable as substances stimulating B-lymphocytes with different efficacy in normal persons and CLL patients. Both collectives showed the best results in the T-lymphocyte culture after stimulation with LPS.     

Isolation, identification and electrophoretic properties of DNA excreted by human lymphocytes

It was shown that the buoyant density of DNA isolated from lymphocyte incubation media as well as from blood plasma of healthy patients and patients with chronic lympholeukemia (CLL) using the SDS-phenol method with subsequent ultracentrifugation in a cesium trifluoroacetate density gradient is 1.59-1.60 and 1.60-1.63 g/ml, respectively. Using electrophoresis in 0.6% agarose gel, it was found that the DNA excreted by lymphocytes from healthy patients and CLL patients is a high molecular weight fraction comprising 21,000 nucleotide pairs. This DNA fragment contains 90-95% of [3H]thymidine used for the labeling of DNA excreted by lymphocytes from healthy patients and patients with CLL. No traces of [3H]thymidine were found in the middle part of the agarose gel containing the diffuse material bound to ethidium bromide.     

Normal B cells express 51p1-encoded Ig heavy chains that are distinct from those expressed by chronic lymphocytic leukemia B cells

51p1 is an allele of V(H)1-69 that frequently is expressed by chronic lymphocytic leukemia (CLL) B cells with little or no somatic mutation. The rearranged 51p1 genes expressed by CLL B cells have a distinctive use of D segments D3-3/DXP4 and D3-10/DXP'1, a favored use of J(H)6, and a longer third complementarity-determining region than the rearranged Ig genes used by CLL B cells that express V(H)1 genes other than V(H)1-69. We examined the 51p1-encoded Ig expressed by blood B cells of healthy donors. In contrast to the infrequent use of J(H)4 by 51p1-expressing CLL (e.g., 4%), 36% of the rearranged 51p1 sequences from normal blood B cells used J(H)4. Furthermore, the D segment use of the rearranged 51p1 sequences from normal blood B cells was not restricted, but reflected the D segment use of nonselected IgH of normal B cells. Finally, the mean length of the third complementarity-determining region for the 51p1 genes of normal blood B cells was 14.6 +/- 4.3 (SD) codons. This is significantly shorter than that noted for 51p1-expressing CLL B cells (18.8 +/- 3.2; p < 0.0001, n = 51). This study demonstrates that the 51p1-encoded IgH expressed in CLL are not representative of the 51p1-encoded IgH expressed by normal blood B cells, indicating that CLL B cells express IgH that are distinctive from those found in the normal adult blood B cell repertoire.     

Immunomodulation by neutrophil myeloperoxidase and hydrogen peroxide: differential susceptibility of human lymphocyte functions

The coexistence of activated polymorphonuclear leukocytes and lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. To test this hypothesis, we examined the effects of neutrophil myeloperoxidase and H2O2 on lymphocytes. Human peripheral blood mononuclear leukocytes were exposed to myeloperoxidase, an H2O2-generating system (glucose + glucose oxidase), and a halide, and were then tested for functional activities. Natural killer activity against K562 cells, lymphocyte proliferation in response to mitogens, and generation of immunoglobulin-secreting cells were all susceptible to oxidative injury by myeloperoxidase and H2O2. The degree as well as the mechanism of suppression was dependent on the glucose oxidase concentration (i.e., the rate of H2O2 delivery). At low H2O2 flux, myeloperoxidase was essential for induction of lymphocyte suppression; as the rate of H2O2 generation increased, suppression became myeloperoxidase-independent and was mediated by H2O2 alone. Various lymphocyte functions were differentially susceptible to oxidative injury by myeloperoxidase and H2O2. The proliferative response to poke-weed mitogen was the least sensitive, whereas antibody formation was the most sensitive. Proliferative responses to concanavalin A and phytohemagglutinin as well as natural killer activity displayed intermediate degrees of susceptibility. In all assays, lymphocyte viability was greater than 90%. Removal of monocytes from mononuclear leukocytes by adherence to glass increased susceptibility of lymphocytes to oxidative injury. Monocytes in proportions within the range present in peripheral blood mononuclear leukocytes protected lymphocyte functions against oxidative injury by myeloperoxidase and H2O2. This study demonstrates a differential susceptibility of various immune functions to oxidative injury by the neutrophil products myeloperoxidase and H2O2, and shows, in addition, that monocytes can modulate these interactions.     

Mechanism of induction of mouse erythrocyte receptor switch in human B cells

An early event in phorbol ester-induced maturation of chronic lymphocytic leukemic (CLL) B cells is a membrane change characterized by the inactivation of a mouse erythrocyte receptor (MER). This event, the MER-switch, is quantified by inhibition of rosette formation. By using [3H]phorbol dibutyrate ([3H]PDBu), both to stimulate MER-switch and assay binding of PDBu to CLL cells, it was shown that MER-switch was an irreversible, time-dependent event which occurred some time after maximal binding of [3H]PDBu to cells. Two classes of binding sites, one of high affinity (Kd 1 to 2 nM) at low frequency (1.5 to 5 X 10(4) sites per cell), and a lower affinity site (Kd 33 to 50 nM) of higher frequency (2 to 3.5 X 10(5) sites per cell), were detected. Binding of [3H]PDBu was inhibited by phorbol ester analogs that stimulated MER-switch, but not by inactive analogs. This, and the similarity in shapes of the binding and rosette inhibition curves over a range of concentrations, suggests that stimulation of MER-switch by phorbol esters is due to this specific binding. The phorbol ester receptor and MER are distinct because MER-ve T cells and MER-ve atypical B cells from a patient with CLL had both classes of PDBu receptor. Solubilized MER did not bind [3H]PDBu. Time-course studies, and the irreversibility of the switch, despite removal of most of the bound [3H]PDBu, indicate that inhibition of rosetting is not due to competitive or steric hindrance by phorbol esters. Equivalent activities of soluble MER were released from fresh and phorbol ester-treated CLL cells, indicating a rearrangement of MER, rather than a loss. A supernatant of phytohemagglutinin-stimulated human spleen cells also induced MER-switch in CLL lymphocytes, suggesting that a lymphokine may be a natural inducer of this event.     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

Lymphocyte monovalent cation metabolism: cell volume, cation content and cation transport

Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm²/min × 10^?8) than in CLL (3.24 ± 0.45 mmoles/cm²/min × 10^?8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10^?3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ～ 20:1, Na ～ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.     

Eph-ephrin bidirectional signaling comes into the context of lymphocyte transendothelial migration

A better knowledge of the molecular mechanisms that govern leukocyte trafficking is of major relevance for the clinics. Both normal and pathologic extravasation of lymphocytes are a fine-tuned spatio-temporal event of migratory path-finding, likely regulated by molecular guidance cues underlying cell movements in other systems. We have recently reported that members of the Eph family of receptor tyrosine kinases, namely EphA2 and one of its ligands, ephrin-A4 (EFNA4) can mediate in the traffic of chronic lymphocytic leukemia (CLL) cells and presumably of normal B cells between the blood and the tissues. The importance of EphA2-EFNA4 interactions at the endothelium-lymphocyte interface during TEM could rely on their attractive/repulsive properties. In the present work, we expand on those results by including additional insights and new suggestions for future studies that discuss the relevance of these molecules in overall cell adhesion dynamic events.Key words: Eph, ephrin, migration pathfinding, trafficking, leukemia, endothelium, lymphocyte, CLL, extravasation, transendothelial migration     

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.