B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

Download Full-text

B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Blood ◽

10.1182/blood.v73.7.1814.1814 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1814-1820 ◽

Cited By ~ 15

Author(s):

L Feldman ◽

N Dainiak

Keyword(s):

Growth Factors ◽

T Lymphocytes ◽

B Lymphocytes ◽

Plasma Membranes ◽

B Lymphocyte ◽

Temperature Stability ◽

Hematopoietic Growth Factors ◽

Serum Free ◽

Gm Csf ◽

Lineage Specificity

Abstract Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

Download Full-text

T lymphocyte-dependent B lymphocyte proliferative response to antigen. I Genetic restriction of the stimulation of B lymphocyte proliferation.

Journal of Experimental Medicine ◽

10.1084/jem.153.4.871 ◽

1981 ◽

Vol 153 (4) ◽

pp. 871-882 ◽

Cited By ~ 20

Author(s):

H Y Tse ◽

J J Mond ◽

W E Paul

Keyword(s):

T Lymphocytes ◽

B Lymphocytes ◽

T Lymphocyte ◽

Lymphocyte Proliferation ◽

B Lymphocyte ◽

Antigen Specificity ◽

Apparent Lack ◽

The Face ◽

Stimulation Of

For the purpose of examining more closely the interaction between T and B lymphocytes, we have developed an in vitro T lymphocyte-dependent B lymphocyte proliferation assay. Proliferation of B lymphocytes in response to antigen was found to depend on the presence of primed T lymphocytes; the B lymphocytes could be derived from nonprimed animals. It appears that these B cells were nonspecifically recruited to proliferate. This nonspecific recruitment, however, was found to be Ir-gene restricted in that B lymphocytes from B10.S mice, which are genetic nonresponders to the polymer Glu60-Ala30-Tyr10 (GAT), could not be stimulated by GAT-primed (responder X nonresponder) F1 T cells. The apparent lack of antigen specificity in the face of Ir gene-restricted T-B interaction may have important implications in our understanding of the recognition unit(s) on T lymphocytes.

Download Full-text

Bone modulation in sustained hematopoietic stimulation in mice

Blood ◽

10.1182/blood.v77.10.2135.2135 ◽

1991 ◽

Vol 77 (10) ◽

pp. 2135-2141 ◽

Cited By ~ 45

Author(s):

MY Lee ◽

R Fukunaga ◽

TJ Lee ◽

JL Lottsfeldt ◽

S Nagata

Keyword(s):

Growth Factors ◽

Bone Resorption ◽

Animal Studies ◽

Peritoneal Macrophages ◽

Hematopoietic Growth Factors ◽

Skeletal Tissue ◽

Gm Csf ◽

Human Erythropoietin ◽

Bone Changes

Abstract To understand the etiology of bone modulation and hypercalcemia observed in granulocytosis of a tumor-bearing animal model and to gain insight into the implication of sustained hematopoietic stimulation on the bone tissue, in vivo responses of normal mouse hematopoietic and bone tissues to long-term injections of recombinant human and murine granulocyte colony-stimulating factor (G-CSF), murine granulocyte- macrophage CSF (GM-CSF), and human erythropoietin were quantitatively analyzed. Osteoclast activation was estimated by the osteoclast- endosteal ratio, determined by morphometric analyses of femoral sections. Medullary and bone areas were measured on transverse ground bone sections of the tibia. Recombinant murine G-CSF provoked marked granulocytosis associated with significant increases in the number of marrow granulocytes and their progenitors, and caused expansion of granulopoietic marrow into fatty marrow. The bone of G-CSF-treated mice showed a significant increase in endosteal osteoclast numbers with medullary area enlargement and a reduction in the bone thickness; indicative of endosteal bone resorption. Although GM-CSF had little effect on granulopoiesis, it caused peritoneal macrophages to increase and induced similar bone changes as those observed in G-CSF treatment. Enhanced erythropoiesis stimulated by erythropoietin was also associated with evidence of endosteal bone resorption. Bone changes induced by these growth factors were not associated with hypercalcemia. These animal studies document association of bone modulation in sustained stimulation of hematopoiesis, and implicate important physiologic effects of hematopoietic growth factors on skeletal tissue in vivo.

Download Full-text

Development of suppressor T lymphocytes for Epstein-Barr virus-induced B-lymphocyte outgrowth during acute infectious mononucleosis: assessment by two quantitative systems

Blood ◽

10.1182/blood.v57.3.510.510 ◽

1981 ◽

Vol 57 (3) ◽

pp. 510-517 ◽

Cited By ~ 20

Author(s):

RT Schooley ◽

BF Haynes ◽

J Grouse ◽

C Payling-Wright ◽

AS Fauci ◽

...

Keyword(s):

T Lymphocytes ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

B Lymphocyte ◽

Acute Stage ◽

Cell Mediated Immunity ◽

Barr Virus ◽

Epstein Barr

Abstract A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

Download Full-text

In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽

10.1182/blood.v78.12.3155.3155 ◽

1991 ◽

Vol 78 (12) ◽

pp. 3155-3161 ◽

Cited By ~ 1

Author(s):

RM Schwartz ◽

SG Emerson ◽

MF Clarke ◽

BO Palsson

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Culture Medium ◽

Culture Conditions ◽

Hematopoietic Growth Factors ◽

Hematopoietic Stem ◽

Gm Csf ◽

Medium Exchange ◽

Proliferation And Differentiation

Abstract We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

Download Full-text

Murine myeloid cells transformed by myb require fibroblast-derived or autocrine growth factors in addition to granulocyte-macrophage colony- stimulating factor for proliferation

Blood ◽

10.1182/blood.v83.1.209.209 ◽

1994 ◽

Vol 83 (1) ◽

pp. 209-216 ◽

Cited By ~ 5

Author(s):

EM Macmillan ◽

TJ Gonda

Keyword(s):

Growth Factors ◽

Myeloid Cells ◽

Colony Stimulating Factor ◽

Hematopoietic Growth Factors ◽

Autocrine Growth ◽

Gm Csf ◽

Absolute Dependence ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Murine myeloid cells can be transformed in vitro by infection with recombinant retroviruses carrying activated myb genes. While these myb- transformed hematopoietic cells (MTHCs) can proliferate continuously in culture, they exhibit several characteristics of progenitor cells of the granulocyte-macrophage (GM) lineage, including an absolute dependence on hematopoietic growth factors (HGFs) such as GM colony- stimulating factor (GM-CSF) for survival and growth. Whereas we have previously shown that MTHCs respond synergistically to certain combinations of HGFs, we report here that MTHCs apparently require two HGFs for proliferation, because GM-CSF alone appears insufficient to promote growth when MTHCs are cultured at very low densities. However, proliferation can be stimulated by either increasing the density at which MTHCs are cultured (implying the production of an autocrine growth factor) or by the presence of a feeder layer of irradiated fibroblasts. We find that the activity of such feeder layers is greatest when the MTHCs are allowed to contact them directly; and by using mutant fibroblast lines, that it depends on the production of CSF- 1, but not Steel factor (SLF). In contrast, the autocrine factor appears not to be either CSF-1 or SLF, and the possibility is raised that it may represent a novel HGF activity. Potential implications of these results for normal and leukemic hematopoiesis are discussed.

Download Full-text

Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽

10.1182/blood.v80.3.670.670 ◽

1992 ◽

Vol 80 (3) ◽

pp. 670-677 ◽

Cited By ~ 45

Author(s):

WJ Murphy ◽

JR Keller ◽

CL Harrison ◽

HA Young ◽

DL Longo

Keyword(s):

Growth Factors ◽

Nk Cells ◽

Nk Cell ◽

Interleukin 2 ◽

Hematopoietic Growth Factors ◽

Ifn Gamma ◽

Gm Csf ◽

Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

Download Full-text

Effect of recombinant hematopoietic growth factors on proliferation of human marrow progenitor cells in serum-deprived liquid culture

Blood ◽

10.1182/blood.v72.4.1387.1387 ◽

1988 ◽

Vol 72 (4) ◽

pp. 1387-1392 ◽

Cited By ~ 18

Author(s):

AR Migliaccio ◽

G Migliaccio ◽

JW Adamson

Keyword(s):

Growth Factors ◽

T Cell ◽

Progenitor Cells ◽

Liquid Culture ◽

Adherent Cell ◽

Hematopoietic Growth Factors ◽

Gm Csf ◽

Normal Human ◽

Fetal Bovine ◽

Marrow Cells

Abstract We investigated the effects of recombinant interleukin-3 (IL-3), granulocyte-macrophage and granulocyte colony-stimulating factors (GM- CSF and G-CSF), and erythropoietin (Ep) on the number of human hematopoietic progenitors after two to ten days of incubation in liquid cultures deprived of fetal bovine serum (FBS). The source of progenitor cells was normal human marrow depleted of T lymphocytes and/or adherent cells. When adherent cell-depleted marrow was cultured without growth factors, the number of progenitor cells was relatively constant for periods up to eight days. In contrast, a progressive decline in the number of progenitor cells was detected in cultures of nonadherent, T- cell-depleted marrow cells. In both cases, the addition of IL-3 increased by two- to fourfold over input the number of erythroid burst- forming cells (BFU-E) per culture. The number of BFU-E peaked either at day 4 or 8. G-CSF had no effect on the number of progenitor cells per culture. GM-CSF and Ep had no effect in cultures of nonadherent marrow cells but maintained the number of BFU-E in cultures of nonadherent, T- cell-depleted marrow cells. The addition of a neutralizing anti-GM-CSF monoclonal antibody, but not anti-IL-3 neutralizing antiserum, decreased the number of BFU-E in cultures of nonadherent marrow cells. None of the growth factors investigated enhanced the number of GM progenitors to the same degree as the number of BFU-E. However, in cultures of nonadherent, T-cell-depleted marrow cells, IL-3 and GM-CSF maintained the number of GM progenitors up to eight days. These results indicate that IL-3 alone is capable of increasing the number of BFU-E and of maintaining the number of GM progenitors in liquid culture, whereas GM-CSF and Ep are capable of maintaining, but not increasing, BFU-E in this system.

Download Full-text

Pre-Stimulation of CML CD34+ Cells with High Concentrations of Growth Factors Counters Imatinib-Mediated Proliferation Suppression and Enhances Apoptosis of Non-Dividing Cells.

Blood ◽

10.1182/blood.v106.11.2479.2479 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2479-2479

Author(s):

Melissa S. Holtz ◽

Stephen J. Forman ◽

Ravi Bhatia

Keyword(s):

Growth Factors ◽

Imatinib Treatment ◽

Hematopoietic Growth Factors ◽

Inhibition Of Proliferation ◽

Gm Csf ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Dividing Cells ◽

High Concentrations

Abstract While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p<0.0001) and 7±3% in high GF (p=0.064)]. However, although overall apoptosis decreased, pre-stimulation resulted in increased apoptosis of undivided cells exposed to imatinib in either low GF (14±5%; p=0.022) or high GF (13±6%, p=0.065). These results are notable since increased apoptosis of undivided cells was not previously observed in any other condition. Importantly, the percent of input cells remaining viable and undivided decreased significantly for pre-stimulated cells exposed to imatinib in low GF (19±3%; p<0.0001) or high GF (7±2%; p=0.016). These results highlight the potential use of GF stimulation to enhance targeting of CML HSC. Additional studies examined whether GF readily available for clinical use (G-CSF and/or GM-CSF) could also enhance imatinib targeting of quiescent CML progenitors. CML CD34+ cells were exposed to 1mM imatinib for 96 hours in a basal low GF cocktail (250pg/ml G-CSF, 10pg/ml GM-CSF, 200pg/ml SCF, 1ng/ml IL6, 200pg/ml MIP1α, 50pg/ml LIF) alone or with the addition of G-CSF (50ng/ml) and/or GM-CSF (10ng/ml). While overall apoptosis decreased, apoptosis of undivided cells significantly increased for cells exposed to imatinib in high concentrations of G-CSF + GM-CSF compared to those in basal GF alone (8.7±1.3%; p=0.007). The percent of input cells remaining viable and undivided in the presence of imatinib significantly decreased with high G-CSF + GM-CSF compared to basal GF alone (7.2%±1.1; p=0.007). In conclusion, pre-stimulation with high concentrations of GF can lead to increased proliferation and enhance reduction of non-dividing CML CD34+ cells by imatinib. These results are of significance because non-dividing primitive cells have previously proven highly resistant to elimination by imatinib and support translational clinical studies to investigate whether intermittent GF administration can enhance elimination of residual CML stem and progenitor cells in patients in remission on imatinib treatment.

Download Full-text

CD39/NTPDase-1 Expression Is Associated with Activation in T-Lymphocytes.

Blood ◽

10.1182/blood.v108.11.1733.1733 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1733-1733

Author(s):

Dianne Pulte ◽

Marinus Johan Broekman ◽

Joan Drosopoulos ◽

Kim E. Olson ◽

Naziba Islam ◽

...

Keyword(s):

T Lymphocytes ◽

Enzymatic Activity ◽

B Lymphocytes ◽

T Lymphocyte ◽

B Lymphocyte ◽

Inflammatory Responses ◽

Geometric Mean ◽

Compensatory Mechanism ◽

Nucleotidase Activity ◽

Cd8 Cells

Abstract CD39/NTPDase-1 is an ecto-ATP/ADPase expressed on leukocytes and endothelial cells. CD39 is the main control system for blood fluidity. CD39 on lymphocytes was first reported in 1991 by Kansas et al. However, studies of CD39 expression and activity on leukocytes have not been done. We characterized levels of CD39 expression and enzymatic activity on neutrophils (PMN), lymphocytes and lymphocyte subsets. Since inflammatory responses occur in arterial vascular disease, we also examined expression of CD39 on naive versus activated and memory lymphocytes. Lymphocytes were isolated by a histopaque procedure, and PMN by dextran gradient. B-lymphocytes were isolated using the RosetteSep B-cell kit. All cell types were confirmed to have purities of >90%. CD39 activity was assayed via our radio-thin-layer chromatographic system. CD39 expression was measured on leukocytes via FACS. PMN, monocytes, and lymphocytes were identified by their forward and side-scatter characteristics. Subsets of lymphocytes were examined via double staining for CD39 and antibodies against specific sub-types. CD39 localized to the surface of greater than 95% of neutrophils, monocytes, and B-lymphocytes. It was also present on a minority (~8%) of T-lymphocytes with no difference in frequency of expression between CD4+ and CD8+ cells. Geometric mean (GM) expression of CD39 per cell was greatest in B-lymphocytes and monocytes, lower in CD4+ cells, and lowest in CD8+ cells and PMN. Interestingly, incubation of T- lymphocytes with PHA up-regulated CD39 in CD8+ cells both in terms of number of cells expressing and GM, with expression rising to 65%. The GM increased 4-fold after 6d of stimulation with PHA. A similar but less dramatic increase was seen with LPS. This is the first time we have accomplished up-regulation of CD39 expression and enzymatic activity. Radio-TLC measurement of nucleotidase activity showed B-lymphocytes>PMN>T-lymphocytes. B-lymphocyte ADPase and ATPase activities (in pmol/min/50K cells) were 75 and 43, respectively. PMN displayed 39 (ADPase) and 22 (ATPase), while T-lymphocytes had enzymatic activity of 16 and 11.5, respectively. ADPase:ATPase ratios were similar for B-lymphocytes and PMN, but lower for T-lymphocytes (1.8 for B-lymphocytes and PMN, vs 1.45 for T-lymphocytes, p=0.03). Lymphocytes stimulated with PHA demonstrated an increase in enzyme activity of 10–20X baseline that peaked at 7–10d. ADPase:ATPase ratio was unchanged. FACS measurement showed that CD39+ lymphocytes were more often activated than CD39− lymphocytes in both CD3+ (p=0.06) and CD4+ (p=0.02) subgroups. Preliminary experiments indicated that >85% of CD39+ T-lymphocytes are CD45RO+. Importantly, this suggests that CD39 is expressed primarily on activated or memory cells in the T-lymphocyte population. Thus, CD39 is expressed on a broad variety of leukocytes. T-lymphocyte expression can be induced by stimulation with mitogens. Moreover, CD39 is present primarily on CD45RO+ T-lymphocytes. We conclude that CD39 expression can be induced by activation of the immune system. The up-regulation of CD39 on activated and memory T-lymphocytes may be a compensatory mechanism for protection from thrombosis as a consequence of inflammation. It may serve as a mechanism for metabolizing extracellular ATP and therefore decreasing the inflammatory stimulus. Abnormalities in CD39 may result in decreased nucleotidase activity and increased vulnerability to thrombosis as a consequence of inflammation.

Download Full-text