Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes

Abstract Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.

Download Full-text

Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes

Blood ◽

10.1182/blood.v81.2.446.bloodjournal812446 ◽

1993 ◽

Vol 81 (2) ◽

pp. 446-453 ◽

Cited By ~ 66

Author(s):

EA Turley ◽

AJ Belch ◽

S Poppema ◽

LM Pilarski

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Transient Expression ◽

Peripheral Blood ◽

Pokeweed Mitogen ◽

Lymphoid Tissues ◽

Hairy Cells ◽

Hodgkin's Lymphomas

Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.

Download Full-text

Plasma and Intracellular Concentration of TNF and Its Receptors in Peripheral Blood and Bone Marrow in Different Stages of B-CLL.

Blood ◽

10.1182/blood.v104.11.4781.4781 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4781-4781

Author(s):

Jacek Rolinski ◽

Agnieszka Bojarska-Junak ◽

Iwona Hus ◽

Anna Dmoszynska

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Lymphocytes ◽

Peripheral Blood ◽

Biological Effects ◽

Leukemic Cells ◽

Control Group ◽

Stage Of Disease ◽

Positive Correlation

Abstract TNF has been proposed to play a role in the regulation of growth and death of leukemic B-CLL cells. However, the biological effects of TNF on leukemic cells, as well as its role as a prognostic factor need to be further investigated. The aim of the study was to eevaluate the correlation of TNF and its receptors in peripheral blood (PB) and bone marrow (BM) with the stage of B-CLL and some other clinical parameters. PB and BM were taken from 44 newly diagnosed, untreated B-CLL. patients. The control group consisted of 20 healthy subjects. We used flow cytometry technique to assess the capability of T and B lymphocytes to produce TNF and ELISA method to measure plasma levels of TNF and their soluble receptors. We found, that PB and BM plasma TNF concentration in the patients was significantly higher than in the healthy control (2.61 pg/ml. vs 0.62 pg/ml; and 2.91 pg/ml vs 0.75 pg/ml, respectively p<0.001). TNF concentration in PB and BM was significantly higher in Rai stage III–IV than in early stages (p<0.01). There was a correlation between the PB and BM TNF level and lymphocytosis (p<0.005) and the total tumor mass (TTM) (p<0.0001). The PB and BM TNF concentration positively correlated with the percentage of T CD3+ lymphocytes producing intracellular TNF (p<0.01). The percentage of T cells from PB an BM expressing cytoplasmic TNF was significantly higher in patients (PB:39.11±16.97%; BM:40.73±18.19%) than in normal controls (PB:15.74±7.95%; BM:18.80±12.93%) (p< 0.00001; p<0.005, respectively). In PB and BM from B-CLL patients the percentage of CD3+ cells expressing intracellular TNF was significantly higher than the percentage of CD19+/TNF+ cells (p<0.0001). Besides, it was found that the percentage of T cells expressing cytoplasmic TNF positively correlated with the stage of disease (p<0.01). In PB positive correlation were found between the number of T CD3+/TNF+ cells and lymphocytosis (p<0.05) and TTM (p<0.001). The percentage of leukaemic B cells positive for TNF did not correlate with the stage of disease. There was increased expression of TNF-RI and TNF-RII in leukaemic B cells in comparison to normal B-cells was observed (p<0.0001). We found positive correlation between the number of CD5+ B lymphocytes and the levels of soluble TNF-RII (sTNF-RII) (p< 0.05). The sTNF-RII levels in PB and BM significantly correlated with the stage of disease acc. Rai (p<0.0001). Furthermore, the sTNF-RII concentration positively correlated with lymphocytosis and TTM (p<0.0001). These results strongly support the key role TNF in B-CLL pathogenesis. Our results suggest that TNF may function as growth factor for B-CLL cells. CD3+T cells may be the important source of this cytokine in advanced B-CLL. It seems that changes in T cells capability to produce cytoplasmic TNF are associated with disease progression. However, further studies are required to confirm the key role of TNF in B-CLL pathogenesis.

Download Full-text

Flow cytometric detection of aneuploid CD38++ plasmacells and CD19+ B-lymphocytes in bone marrow, peripheral blood and PBSC harvest in multiple myeloma patients

Leukemia Research ◽

10.1016/j.leukres.2003.09.015 ◽

2004 ◽

Vol 28 (5) ◽

pp. 469-477 ◽

Cited By ~ 38

Author(s):

Anna Maria Santonocito ◽

Ugo Consoli ◽

Sabrina Bagnato ◽

Giuseppe Milone ◽

Giuseppe A. Palumbo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Lymphocytes ◽

Peripheral Blood ◽

Flow Cytometric

Download Full-text

Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽

10.1182/blood.v73.7.1925.bloodjournal7371925 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1925-1935

Author(s):

MA King ◽

DS Nelson

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Bone Marrow Cells ◽

Cell Types ◽

Pokeweed Mitogen

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Download Full-text

Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽

10.1182/blood.v73.7.1925.1925 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1925-1935 ◽

Cited By ~ 32

Author(s):

MA King ◽

DS Nelson

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Bone Marrow Cells ◽

Cell Types ◽

Pokeweed Mitogen

Abstract Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Download Full-text

Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas.

Journal of Experimental Medicine ◽

10.1084/jem.178.3.1091 ◽

1993 ◽

Vol 178 (3) ◽

pp. 1091-1096 ◽

Cited By ~ 80

Author(s):

P Corradini ◽

M Boccadoro ◽

C Voena ◽

A Pileri

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Plasma Cell ◽

Plasma Cells ◽

Bone Marrow Cells ◽

Indirect Evidence ◽

Specific Marker

Multiple myeloma is a B cell malignancy characterized by the expansion of plasma cells producing monoclonal immunoglobulins (Ig). It has been regarded as a tumor arising at the B, pre-B lymphocyte, or even stem cell level. Precursor cells are presumed to proliferate and differentiate giving rise to the plasma cell clonal expansion. Antigenic features and specific Ig gene rearrangement shared by B lymphocytes and myeloma cells have supported this hypothesis. However, the existence of such a precursor is based upon indirect evidence and is still an open question. During differentiation, B cells rearrange variable (V) regions of Ig heavy chain genes, providing a specific marker of clonality. Using an anchor polymerase chain reaction assay, these rearranged regions from five patients with multiple myeloma were cloned and sequenced. The switch of the Ig constant (C) region was used to define the B cell differentiation stage: V regions are linked to C mu genes in pre-B and B lymphocytes (pre-switch B cells), but to C gamma or C alpha in post-switch B lymphocytes and plasma cells (post-switch B cells). Analysis of bone marrow cells at diagnosis revealed the presence of pre-switch B cells bearing plasma cell V regions still joined to the C mu gene. These cells were not identified in peripheral blood, where tumor post-switch B cells were detected. These pre-switch B cells may be regarded as potential myeloma cell precursors.

Download Full-text

A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽

10.1182/blood.v84.6.1922.1922 ◽

1994 ◽

Vol 84 (6) ◽

pp. 1922-1930 ◽

Cited By ~ 142

Author(s):

T Goto ◽

SJ Kennel ◽

M Abe ◽

M Takishita ◽

M Kosaka ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Affinity Constant ◽

Target Antigen ◽

Specific Marker

Abstract A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

Download Full-text

Studies on the Tissue-Related Phenotypic Heterogeneity of Murine B Cells

Developmental Immunology ◽

10.1155/1998/61492 ◽

1998 ◽

Vol 6 (3-4) ◽

pp. 179-185 ◽

Cited By ~ 1

Author(s):

Péter Balogh ◽

Attila Kumánovics ◽

Istvan Juhasz

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Phenotypic Heterogeneity ◽

Multiparameter Flow Cytometry ◽

Lymphoid Tissues ◽

Altered Expression ◽

Significant Difference ◽

B Cell Precursors ◽

The Relationship

The development of B cells is accompanied by their ability to specifically enter the peripheral lymphoid tissues. Recently, we described a novel rat monoclonal antibody (IBL-2; IgG2b/kreacting with a 26/29-kD heterodimeric structure of the cell surface. This mAb has been found to recognize differentially the peripheral B cells of mice depending on their tissue origin. The majority of splenic B cells as well as the mature B cells in the bone marrow were stained with this mAb, whereas the B lymphocytes isolated from LN or Peyer's patches displayed only negligible reactivity. We extended these observations by analyzing the relationship between the expression of IBL-2 antigen and L-selection on the surface of B-cell precursors in the bone marrow by multiparameter flow cytometry. Within the B220 positive compartment, a significant difference of L-selectin expression could be observed between the various IBL-2-reactive subsets. Furthermore, we investigated whether evidences for the establishment of tissue-associated phenotypic heterogeneity similar to that found in normal mice could be found upon the adoptive transfer of normal unselected splenic lymphocytes into SCID recipients (Spl-SCID). It has been found that a large part of the splenic B cells preserved their IBL-2 reactivity, whereas the LN B cells had lost the IBL-2 antigen in Spl-SCID. These data indicate that the phenotypic difference within the SCID mice may be the result of the migration of B lymphocytes from the spleen toward the lymph nodes, and the altered expression of the IBL-2 antigen correlates with this process.

Download Full-text

A novel membrane antigen selectively expressed on terminally differentiated human B cells

Blood ◽

10.1182/blood.v84.6.1922.bloodjournal8461922 ◽

1994 ◽

Vol 84 (6) ◽

pp. 1922-1930 ◽

Cited By ~ 1

Author(s):

T Goto ◽

SJ Kennel ◽

M Abe ◽

M Takishita ◽

M Kosaka ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Affinity Constant ◽

Target Antigen ◽

Specific Marker

A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.

Download Full-text

Circulating B Lymphocytes from Patients with Multiple Myeloma Harbour T(4;14) Translocations and Chromosome 13 Deletions.

Blood ◽

10.1182/blood.v106.11.500.500 ◽

2005 ◽

Vol 106 (11) ◽

pp. 500-500 ◽

Cited By ~ 1

Author(s):

Carina Debes-Marun ◽

Andrew R. Belch ◽

Linda M. Pilarski

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Chromosomal Abnormalities ◽

Minority Population ◽

Polymorphonuclear Cells ◽

Chromosome 13 ◽

Healthy Donors

Abstract Multiple Myeloma (MM) is clinically characterized by accumulations of plasma cells in the bone marrow (BM). We have identified drug resistant clonotypic B lymphocytes in the peripheral blood of MM patients that have malignant characteristics and the ability to xenograft MM to immunodeficient mice. Using an automated scanning system (Bioview Duet), for blood samples from 72 MM patients, we scanned cytospin slides stained with May-Grunwald Giemsa to identify lymphocytes and determine whether they have the same chromosomal abnormalities that characterize autologous plasma cells. The location on the slide of each morphologically identified cell is recorded during an initial scan, followed by FISH, and examination of the same cells for genetic abnormalities. For this study, we chose probes to detect 1) deletion of chromosome 13 using D13S319 (Vysis), and 2) the t(4;14)(p16;q32) translocation using a dual fusion probe (Vysis). Both abnormalities correlate with adverse prognosis. Lymphocytes from PBMC of healthy donors show 2.8+/−1% of lymphocytes with Ch13 deletion, and less than 1% with t(4;14). An MM PBMC sample was considered to have lymphocyte abnormalities if it scored above a cut off value of 10% for Ch13 deletions and 2% for t(4;14), this is likely to underestimate the extent of chromosomal abnormalities in MM B cells. The number of abnormal lymphocytes in MM PBMC is sufficiently large, and the number of abnormal cells in comparable populations from healthy donors is sufficiently small, that we are readily able to detect significantly increased numbers of abnormal lymphocytes in MM PBMC. We found that peripheral lymphocytes from 19/60 (32%) MM patients have Ch13 deletion (27%) and/or t(4;14) (26%); for those patients with an available BM sample, these same abnormalities were found in their BM plasma cells. Interestingly, intraclonal heterogeneity is apparent in malignant cells from a t(4;14) patient; these plasma cells include a minority population with apparent Ch14 monosomy (23%) and a major population having two copies of Ch14 (77%). This was internally controlled in that for both populations, the plasma cells have two copies of the fused t(4;14) chromosome, and the polymorphonuclear cells on the same slide were normal. One MM patient had Ch13 deletion in 60% of lymphocytes from PBMC, consistent with the 63% of BM plasma cells from this same patient showing Ch13 deletion. For another MM patient known to be t(4;14)+, sorted sIgM+ B cells were analyzed and found to include a small subset with t(4;14) translocations. For 2/6 MM PBMC, abnormalities in Ch19 were detectable in 25–37% of lymphocytes. For mobilized blood autografts, preliminary data indicates the presence of lymphocytic cells with detectable Ch13 deletion. By performing FISH after immunostaining with anti-CD20 of PBMC from MM patients whose bone marrow plasma cells had Ch13 deletion/monosomy, we detected chromosomal abnormalities in CD20+ B cells from MM blood. No abnormalities were detected for CD20+ B cells from healthy donors. In the context of our previous work, this analysis demonstrates that two molecular signatures of MM, clonotypic IgH gene rearrangements and, as reported here, prognostically important chromosomal abnormalities are found among circulating CD20+ B cells from the blood of patients with MM. This work confirms that the MM clone includes circulating B lymphocytes harbouring chromosomal deletions or IgH switch region translocations known to be clinically significant in MM, further implicating them as a source of relapse.

Download Full-text